{"gene":"CHAC1","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":2012,"finding":"ChaC1 and its homologues function as γ-glutamyl cyclotransferases that specifically degrade glutathione (but not other γ-glutamyl peptides) both in vivo and in vitro; catalytically dead E>Q mutants failed to deplete glutathione or enhance apoptosis in yeast, establishing enzymatic activity as required for these effects.","method":"In vitro enzymatic assays, yeast overexpression with catalytic mutants, glutathione depletion measurements","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution of enzymatic activity combined with active-site mutagenesis and in vivo validation in yeast; foundational mechanistic paper replicated by subsequent studies","pmids":["23070364"],"is_preprint":false},{"year":2009,"finding":"CHAC1 is a downstream component of the ATF4-ATF3-CHOP arm of the unfolded protein response (UPR); it is not activated by the parallel XBP1 or ATF6 branches. CHAC1 overexpression enhanced apoptosis and its knockdown suppressed apoptosis (TUNEL, PARP cleavage, AIF nuclear translocation), placing it as a proapoptotic effector downstream of ATF4.","method":"siRNA knockdown of ATF4/ATF3/CHOP/XBP1/ATF6, CHAC1 overexpression plasmids, TUNEL assay, PARP cleavage, AIF nuclear translocation, expression microarray","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal loss-of-function and gain-of-function experiments with defined pathway placement; independently replicated in many subsequent studies","pmids":["19109178"],"is_preprint":false},{"year":2015,"finding":"Human CHAC1 promoter is transactivated by ATF4 and ATF3 through two cis-elements: a -267 ATF/CRE site and a novel -248 ATF/CRE modifier (ACM) element. Direct binding of ATF4 and ATF3 (and C/EBPβ) at these elements was confirmed by EMSA and ChIP. CHAC1 overexpression robustly depleted glutathione in HEK293 cells; a catalytic mutant failed to do so.","method":"Luciferase reporter with promoter deletion/mutation analysis, immunoblot-EMSA, ChIP, glutathione measurement in cells overexpressing WT vs. catalytic mutant CHAC1","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (reporter assays, EMSA, ChIP, mutagenesis, enzymatic readout) in a single rigorous study confirming both transcriptional regulation and catalytic function","pmids":["25931127"],"is_preprint":false},{"year":2016,"finding":"TRIB3 represses CHAC1 expression downstream of ATF4, protecting cells from arsenite-induced glutathione depletion and death. Two regulatory elements in the Chac1 promoter mediate induction by arsenite/ATF4 and suppression by TRIB3. Chac1 silencing in Trib3-deficient cells restored glutathione and rescued arsenite sensitivity, establishing TRIB3 as an upstream negative regulator of CHAC1.","method":"Trib3-knockout MEFs, Chac1 siRNA, promoter analysis, glutathione measurements, cell viability assays","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with KO cells plus promoter dissection and functional rescue, single lab","pmids":["27526673"],"is_preprint":false},{"year":2016,"finding":"In glioblastoma cells, TMZ-induced CHAC1 expression is controlled transcriptionally by the JNK1/c-JUN pathway. CHAC1 binds to the Notch3 protein and inhibits Notch3 activation, thereby attenuating Notch3-mediated downstream signaling; CHAC1 overexpression induced apoptosis via caspase-3/9 activation, ROS generation, increased intracellular calcium, and loss of mitochondrial membrane potential.","method":"Transcriptome microarray, CHAC1 overexpression/knockdown, co-immunoprecipitation (CHAC1–Notch3 binding), caspase activation assays, ROS measurement, mitochondrial membrane potential assay","journal":"Neuropharmacology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP demonstrating CHAC1–Notch3 interaction supported by multiple functional readouts, single lab","pmids":["27986595"],"is_preprint":false},{"year":2017,"finding":"CHAC1 degrades glutathione in TNBC cells under cystine starvation downstream of the GCN2-eIF2α-ATF4 pathway (not PERK). CHAC1 knockdown rescued GSH levels and prevented cystine-starvation-induced necroptosis and ferroptosis, placing CHAC1 as a functional effector of GCN2-driven cell death.","method":"CHAC1 siRNA knockdown, GSH measurement, cell death assays with pathway inhibitors (Nec-1, MLKL inhibitor, deferoxamine, ferrostatin-1), GCN2/PERK pathway analysis","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic loss-of-function with defined biochemical readout (GSH) and pharmacological dissection of cell death pathway, single lab","pmids":["29383104"],"is_preprint":false},{"year":2018,"finding":"cagA-positive H. pylori infection triggers CHAC1 overexpression in gastric epithelial cells, leading to glutathione degradation, ROS accumulation, and TP53 somatic mutations. A catalytically inactive CHAC1 mutant did not induce TP53 mutations, confirming that CHAC1's glutathione-degrading enzymatic activity is necessary for ROS-mediated genotoxicity.","method":"CHAC1 overexpression (WT vs. catalytic mutant), CHAC1 siRNA, TP53 mutation sequencing, glutathione and ROS measurement in AGS cells","journal":"FEBS open bio","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — active-site mutagenesis linking catalytic activity to downstream genotoxic phenotype, single lab","pmids":["29632819"],"is_preprint":false},{"year":2019,"finding":"Zebrafish Chac1 is a glutathione-degrading enzyme (acting only on reduced GSH) that is required for calcium signaling during embryonic development. chac1 morphants showed attenuated calcium transients (measured by GCaMP6s) and developmental defects in myotome, brain, and heart; these phenotypes were rescued by WT Chac1 but not catalytically inactive Chac1. Intracellular glutathione redox potential (monitored by Grx1-roGFP2) is an upstream activator of calcium signaling.","method":"Zebrafish morpholino knockdown, rescue with WT and catalytically inactive Chac1, live calcium imaging (GCaMP6s), glutathione redox sensor (Grx1-roGFP2)","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vivo reconstitution with catalytic mutant rescue, orthogonal live reporters for both redox and calcium, mechanistically rigorous","pmids":["31189567"],"is_preprint":false},{"year":2020,"finding":"CHAC1 transcription in humans and mice is induced by ATF4 through synergistic ATF/CRE and CARE (amino acid response element) sequences in the CHAC1 promoter. CHOP (DDIT3) exerts a novel inhibitory effect on CHAC1 transcription via the ATF/CRE motif through an indirect mechanism; direct binding of CHOP to the ATF/CRE was not detected by EMSA.","method":"Promoter-luciferase reporter assays, deletion/mutation analysis, IM-EMSA for ATF4 and CHOP binding, ATF4 overexpression in human and mouse cells","journal":"Biochemistry and biophysics reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple promoter dissection methods, but CHOP inhibitory mechanism is indirect and not fully resolved; single lab","pmids":["33102815"],"is_preprint":false},{"year":2022,"finding":"Active-site residues of human ChaC1 forming direct interactions with glutathione were identified: Y38, G39, S40, L41 (38-YGSL-41), D68, R72, E115, and Y143. Residues exclusive to the ChaC family (forming the active-site surface) are required for structural stability. Validation was performed by in silico docking, MD simulations, MMGBSA binding energy, and in vivo yeast activity assays with point mutants.","method":"Molecular docking of glutathione with modeled hChaC1, MD simulations, MMGBSA binding energy calculations, yeast in vivo activity assays of site-directed mutants","journal":"Proteins","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — combined computational and experimental (yeast in vivo) validation of active-site residues; in vitro enzymatic reconstitution not directly reported in abstract","pmids":["36456186"],"is_preprint":false},{"year":2022,"finding":"DJ-1 (PARK7) binds the basic leucine zipper domain of ATF3 (shown by bimolecular fluorescence complementation) and inhibits ATF3 binding to the CHAC1 promoter, thereby downregulating CHAC1 expression and reducing glutathione degradation in astrocytes. DJ-1 knockout significantly upregulated CHAC1.","method":"DJ-1 knockout, high-throughput sequencing, bimolecular fluorescence complementation (BiFC) for DJ-1–ATF3 interaction, promoter-binding assay, GSH measurement","journal":"Neuroscience research","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — BiFC protein interaction plus KO phenotype linked to CHAC1 expression and GSH levels; single lab","pmids":["35988816"],"is_preprint":false},{"year":2023,"finding":"MIA3 binds directly to CHAC1 protein (co-immunoprecipitation and confocal co-localization) and promotes CHAC1 expression and glutathione degradation, thereby promoting hepatocellular carcinoma cell growth and metastasis. MIA3 knockout reduced CHAC1 expression and slowed GSH degradation.","method":"Co-immunoprecipitation, confocal microscopy, MIA3 overexpression/knockout, RNA-seq, GSH measurement, cell proliferation/migration/invasion assays","journal":"Molecular and cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — reciprocal Co-IP and confocal co-localization establishing protein interaction, linked to enzymatic/functional readout; single lab","pmids":["37948019"],"is_preprint":false},{"year":2023,"finding":"CHAC1 inactivation via CRISPR/Cas9 knock-in of an enzyme-inactivating mutation preserved muscle glutathione levels under fasting, cancer cachexia, and chemotherapy conditions, but failed to prevent muscle wasting, demonstrating that glutathione preservation alone is insufficient to protect against cachexia-induced muscle loss.","method":"CRISPR/Cas9 knock-in of enzymatic-inactivating mutation in mice, glutathione measurement, muscle wasting phenotypic analysis under multiple cachexia models","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vivo catalytic knock-in with direct biochemical (GSH) and phenotypic readout; single lab, but rigorous genetic approach","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, activating glycolysis-related gene expression and the Warburg effect in lung adenocarcinoma cells. E2F Transcription Factor 1 was identified as a transcriptional activator of CHAC1 by directly binding the CHAC1 promoter.","method":"Shotgun mass spectrometry-based proteomics, RNA-seq, co-IP for UBA2-CHAC1-PKM2 complex, SUMOylation assays, nuclear/cytoplasmic fractionation, ChIP for E2F1 at CHAC1 promoter, CHAC1 overexpression/knockdown in vitro and in vivo","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mass spectrometry-identified complex confirmed by Co-IP, with functional SUMOylation and localization readouts; single lab","pmids":["39368995"],"is_preprint":false},{"year":2025,"finding":"ATF4 directly binds the CHAC1 promoter (ChIP-seq and ChIP-qPCR) in renal tubular cells; ATF4 depletion inhibited CHAC1 upregulation and pro-ferroptotic responses induced by oxalate, establishing ATF4 as the direct transcriptional activator of CHAC1 in the ER stress-dependent ferroptosis pathway in calcium oxalate kidney stone models.","method":"ChIP-seq, ChIP-qPCR, ATF4 siRNA knockdown, 4-PBA (ER stress inhibitor) treatment, CHAC1 knockout, GSH/lipid peroxidation/Fe2+ measurements in vivo and in vitro","journal":"Advanced science","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — ChIP-seq/ChIP-qPCR directly demonstrating ATF4 occupancy at CHAC1 promoter, combined with in vivo genetic KO and biochemical ferroptosis readouts","pmids":["39836526"],"is_preprint":false},{"year":2025,"finding":"CHAC1 haploinsufficiency (Chac1+/− mice) conferred resilience to kidney disease (folic acid nephropathy, adenine-induced CKD, diabetic nephropathy). Tubule cells from Chac1+/− mice showed increased glutathione, decreased lipid peroxidation, improved viability, and protection against ferroptosis, establishing CHAC1 as a mediator of kidney disease through glutathione degradation and ferroptosis.","method":"CRISPR-generated Chac1+/− mice, multiple kidney disease models, primary tubule cell isolation, GSH measurement, lipid peroxidation assays, ferroptosis gene expression analysis, correlation with kidney biopsy protein data","journal":"Science translational medicine","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vivo genetic loss-of-function in multiple disease models with direct biochemical readouts (GSH, lipid peroxidation, ferroptosis markers); replicated across multiple model systems","pmids":["40267214"],"is_preprint":false},{"year":2025,"finding":"The transcription factor BACH1 activates the CHAC1 promoter (dual-luciferase assay). BACH1 silencing attenuated ferroptosis by suppressing CHAC1 and restoring the GSH-GPX4 axis in cardiomyocytes. CHAC1 overexpression depleted GSH, suppressed GPX4, and enhanced lipid peroxidation during myocardial ischemia-reperfusion, while CHAC1 knockdown was partially protective; in vivo AAV-mediated CHAC1 overexpression worsened cardiac dysfunction and enlarged infarct area.","method":"Dual-luciferase promoter assay for BACH1-CHAC1, BACH1 siRNA, CHAC1 overexpression/knockdown, AAV-mediated cardiac CHAC1 overexpression in mice, GSH/GPX4/lipid peroxidation assays","journal":"Antioxidants","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — promoter assay plus in vivo AAV and genetic KD with multiple biochemical readouts; single lab","pmids":["41750596"],"is_preprint":false},{"year":2025,"finding":"GDIL lncRNA binds and relocalizes the nuclear exoribonuclease XRN2 to the cytoplasm, where GDIL serves as a scaffold for XRN2 to identify and degrade CHAC1 mRNA, thereby reducing CHAC1 protein levels, boosting GSH, and promoting platinum resistance in colorectal cancer.","method":"RNA pulldown, GDIL-XRN2 interaction assays, metabolomic and metabolic flux analysis, CHAC1 mRNA stability assays, antisense oligonucleotide suppression in cell lines and patient-derived xenografts","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods identifying post-transcriptional regulation of CHAC1 mRNA by XRN2 scaffold; single lab","pmids":["39893168"],"is_preprint":false},{"year":2024,"finding":"m6A methylation by METTL3/14 destabilizes CHAC1 mRNA via YTHDF2-mediated decay (RIP assays confirmed METTL3/YTHDF2-CHAC1 mRNA interaction). Arsenic treatment reduced METTL3/14 expression, inhibited m6A modification of CHAC1 mRNA, and increased CHAC1 mRNA half-life (~2-fold), leading to elevated CHAC1 protein, glutathione degradation, GPX4 suppression, and β-cell ferroptosis. METTL3 overexpression restored CHAC1 mRNA degradation and alleviated arsenic-induced dysfunction.","method":"MeRIP (m6A sequencing), RIP assay, METTL3/14 overexpression, CHAC1 mRNA half-life measurement (actinomycin D), CHAC1 knockdown, GSH/GPX4/ferroptosis assays","journal":"Ecotoxicology and environmental safety","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MeRIP and RIP demonstrating m6A-YTHDF2 regulation of CHAC1 mRNA stability, multiple orthogonal methods, single lab","pmids":["39667319"],"is_preprint":false},{"year":2025,"finding":"NUPR1 physically interacts with ATF4 (co-immunoprecipitation in HK2 cells) and suppresses ferroptosis in renal ischemia-reperfusion injury by inhibiting the ATF4-CHAC1 pathway; NUPR1 overexpression reduced CHAC1 expression while NUPR1 knockdown enhanced ferroptosis via ATF4-CHAC1 activation.","method":"Co-immunoprecipitation (NUPR1–ATF4), NUPR1 overexpression/knockdown, renal IRI mouse model, hypoxia-reoxygenation HK2 cell model, ferroptosis marker measurement","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP establishing NUPR1-ATF4 protein interaction plus in vivo and in vitro functional consequences for CHAC1-dependent ferroptosis; single lab","pmids":["42019644"],"is_preprint":false},{"year":2025,"finding":"H3K9 acetylation at the CHAC1 promoter (mediated by increased histone acetyltransferase P300) is required for DHA-induced transcriptional upregulation of CHAC1 in hepatic stellate cells. ATF4 binds two essential CHAC1 promoter regions (-212 to -199 bp and -269 to -257 bp), confirmed by luciferase reporter assays and ChIP-qPCR; inhibiting histone acetylation or ATF4 both blocked CHAC1 induction and reduced ferroptosis.","method":"ChIP-qPCR for H3K9ac and ATF4, luciferase reporter with WT and mutated CHAC1 promoter, P300 inhibition, ATF4 knockdown, RNA-seq, in vivo CCl4 fibrosis model","journal":"Chinese medical journal","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — ChIP, reporter assays, and in vivo experiments establishing epigenetic (H3K9ac/P300) and ATF4 regulation of CHAC1 transcription; single lab","pmids":["40947783"],"is_preprint":false},{"year":2025,"finding":"EGR1 transcriptionally activates CHAC1 expression; EGR1 binding to the CHAC1 promoter was validated by chromatin immunoprecipitation and dual-luciferase reporter assays. EGR1-driven CHAC1 upregulation promotes ferroptosis in keratinocytes; caffeic acid inhibits ferroptosis by suppressing the EGR1-CHAC1 axis.","method":"ChIP assay, dual-luciferase reporter assay, EGR1/CHAC1 overexpression and knockdown plasmids, ferroptosis markers (MDA, ROS, Fe2+, GSH), flow cytometry","journal":"Current molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP and reporter assays directly demonstrating EGR1 binding and transactivation of CHAC1 promoter; single lab","pmids":["39129295"],"is_preprint":false},{"year":2024,"finding":"Juglone (a naturally occurring naphthoquinone) inhibits ChaC1 enzymatic activity with an IC50 of 8.7 µM in vitro; inhibition is non-competitive with glutathione and occurs through a novel mechanism independent of cysteine adduct formation (a cysteine-free ChaC1 variant was still inhibited). Plumbagin was also identified as an effective inhibitor.","method":"Yeast-based high-throughput functional screens, in vitro ChaC1 enzymatic assays, kinetic studies (competitive vs. non-competitive), cysteine-free ChaC1 variant testing","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic characterization of inhibitor mechanism with genetic variant controls; single lab, peer-reviewed publication","pmids":["39400295"],"is_preprint":false},{"year":2025,"finding":"CHAC1 promotes ferroptosis in sepsis-exposed cardiomyocytes via the ATF4-CHOP-CHAC1 axis. miR-383-3p directly targets ATF4 mRNA (validated by dual-luciferase assay), thereby suppressing the ATF4-CHOP-CHAC1 pathway; ATF4 overexpression abolished miR-383-3p's protective effects, and ATF4 knockdown phenocopied miR-383-3p suppression of CHAC1 and ferroptosis.","method":"Dual-luciferase reporter assay (miR-383-3p–ATF4), ATF4 overexpression/knockdown, LPS cardiomyocyte model, miR-383-3p mimics/inhibitors, in vivo sepsis mouse model, ferroptosis markers","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — luciferase validation of miRNA-ATF4 interaction combined with genetic epistasis placing ATF4 upstream of CHOP-CHAC1; single lab","pmids":["41093083"],"is_preprint":false}],"current_model":"CHAC1 is a cytosolic γ-glutamyl cyclotransferase that specifically degrades glutathione (not other γ-glutamyl peptides), depleting intracellular GSH and thereby promoting oxidative stress, ferroptosis, and apoptosis; its transcription is activated primarily by ATF4 (and ATF3) through defined ATF/CRE and CARE promoter elements downstream of the PERK-eIF2α arm of the UPR, and is further modulated by additional transcription factors (EGR1, BACH1), epigenetic marks (H3K9 acetylation via P300), m6A mRNA modification (METTL3/YTHDF2), and regulatory proteins (TRIB3 as repressor, NUPR1 via ATF4 interaction, DJ-1 via ATF3 binding); beyond GSH degradation, CHAC1 also acts as a scaffold enhancing PKM2 SUMOylation to drive nuclear translocation and the Warburg effect, binds Notch3 to inhibit its signaling, and is required for calcium transient generation during zebrafish development through its ability to alter intracellular redox potential."},"narrative":{"mechanistic_narrative":"CHAC1 is a cytosolic γ-glutamyl cyclotransferase that specifically degrades intracellular reduced glutathione, and through this enzymatic depletion of GSH it drives oxidative stress, ferroptosis, and apoptosis across diverse cell types [PMID:23070364, PMID:31189567, PMID:40267214]. Its substrate specificity is narrow — it acts on glutathione but not other γ-glutamyl peptides — and catalysis depends on a defined active-site surface in which residues such as Y38–L41, D68, R72, E115, and Y143 contact glutathione; catalytically dead mutants fail to deplete GSH or produce downstream phenotypes [PMID:23070364, PMID:36456186]. CHAC1 functions principally as a downstream effector of the integrated stress response: it is a proapoptotic component of the ATF4–ATF3–CHOP arm of the UPR, with ATF4 (and ATF3) directly transactivating the CHAC1 promoter through ATF/CRE and CARE elements [PMID:19109178, PMID:25931127, PMID:39836526], and it operates downstream of both PERK and GCN2 branches depending on the stress, mediating cystine-starvation necroptosis and ferroptosis [PMID:29383104]. In vivo genetic studies establish that CHAC1's GSH-degrading activity is required for pathology: Chac1 haploinsufficiency or catalytic inactivation preserves glutathione, suppresses lipid peroxidation, and confers resistance to ferroptosis-driven kidney disease [PMID:39836526, PMID:40267214], although GSH preservation alone is insufficient to prevent cachexia-induced muscle wasting [PMID:37014882]. Beyond GSH degradation, CHAC1 binds Notch3 and inhibits its activation [PMID:27986595], bridges UBA2 and PKM2 to enhance PKM2 SUMOylation and nuclear translocation driving the Warburg effect [PMID:39368995], and is required for glutathione-redox-dependent calcium transients during zebrafish development [PMID:31189567].","teleology":[{"year":2009,"claim":"Established CHAC1 as a proapoptotic effector positioned specifically within the ATF4-ATF3-CHOP arm of the UPR, distinguishing it from the parallel XBP1/ATF6 branches.","evidence":"siRNA knockdown of UPR transcription factors plus CHAC1 gain/loss-of-function with apoptosis readouts (TUNEL, PARP cleavage, AIF translocation)","pmids":["19109178"],"confidence":"High","gaps":["Molecular activity of CHAC1 not yet defined at this stage","Direct promoter binding not yet demonstrated"]},{"year":2012,"claim":"Defined the molecular function of CHAC1 as a γ-glutamyl cyclotransferase that specifically degrades glutathione, and showed this enzymatic activity is required for its proapoptotic effect.","evidence":"In vitro enzymatic assays and yeast overexpression with catalytic E>Q mutants, glutathione depletion measurements","pmids":["23070364"],"confidence":"High","gaps":["Active-site architecture not resolved","Mammalian in vivo relevance not yet established"]},{"year":2015,"claim":"Identified the cis-regulatory basis of CHAC1 induction, mapping direct ATF4/ATF3 binding to ATF/CRE and ACM promoter elements and confirming enzymatic GSH depletion in human cells.","evidence":"Promoter deletion luciferase reporters, EMSA, ChIP, and WT-vs-catalytic-mutant GSH measurement in HEK293","pmids":["25931127"],"confidence":"High","gaps":["Cell-type-specific cofactors not addressed","Quantitative contribution of each element in vivo unknown"]},{"year":2016,"claim":"Revealed both negative transcriptional control and a non-enzymatic moonlighting function, with TRIB3 repressing CHAC1 and CHAC1 binding Notch3 to inhibit its signaling.","evidence":"Trib3-KO MEFs with promoter analysis and rescue; transcriptome profiling, Co-IP, and apoptosis/ROS/calcium/mitochondrial assays in glioblastoma cells","pmids":["27526673","27986595"],"confidence":"Medium","gaps":["CHAC1-Notch3 interaction from single Co-IP without reciprocal/structural validation","Whether Notch3 inhibition is enzyme-dependent unresolved"]},{"year":2017,"claim":"Extended CHAC1's effector role to a second stress branch, showing it executes GCN2-eIF2α-ATF4-driven necroptosis and ferroptosis under cystine starvation, independent of PERK.","evidence":"CHAC1 siRNA with GSH measurement and pharmacological cell-death dissection (Nec-1, ferrostatin-1, deferoxamine) in TNBC cells","pmids":["29383104"],"confidence":"Medium","gaps":["Single lab","Branch selectivity between PERK and GCN2 not mechanistically explained"]},{"year":2018,"claim":"Demonstrated that CHAC1 enzymatic GSH degradation has genotoxic consequences, linking H. pylori-induced CHAC1 to ROS accumulation and TP53 mutation.","evidence":"WT-vs-catalytic-mutant overexpression, siRNA, TP53 sequencing, GSH/ROS measurement in AGS gastric cells","pmids":["29632819"],"confidence":"Medium","gaps":["Direct link between specific ROS species and mutation spectrum not established","Single lab"]},{"year":2019,"claim":"Established a developmental and physiological role beyond cell death, showing GSH-redox-dependent CHAC1 activity is required for calcium transient generation in vivo.","evidence":"Zebrafish morpholino knockdown with WT vs catalytically-inactive rescue, GCaMP6s calcium imaging, Grx1-roGFP2 redox sensing","pmids":["31189567"],"confidence":"High","gaps":["Molecular link between redox potential and calcium machinery not defined","Mammalian conservation of this role untested"]},{"year":2020,"claim":"Refined transcriptional logic by showing ATF4 acts through synergistic ATF/CRE and CARE elements while CHOP exerts an indirect inhibitory effect, complicating the simple ATF4-CHOP-CHAC1 chain.","evidence":"Promoter-luciferase, deletion/mutation analysis, IM-EMSA for ATF4 and CHOP in human and mouse cells","pmids":["33102815"],"confidence":"Medium","gaps":["Mechanism of indirect CHOP repression unresolved (no direct binding detected)","Single lab"]},{"year":2022,"claim":"Provided structural insight into substrate recognition and uncovered additional transcriptional repression via DJ-1-ATF3 interaction.","evidence":"Molecular docking/MD/MMGBSA with yeast point-mutant validation of active-site residues; BiFC of DJ-1-ATF3 with promoter-binding and GSH assays","pmids":["36456186","35988816"],"confidence":"Medium","gaps":["No experimental crystal/cryo-EM structure","In vitro enzymatic reconstitution of active-site mutants not directly reported"]},{"year":2023,"claim":"Separated CHAC1's enzymatic and disease functions and added a direct protein partner, showing catalytic inactivation preserves muscle GSH yet fails to prevent cachexia, while MIA3 binds and stabilizes CHAC1.","evidence":"CRISPR knock-in of enzyme-inactivating mutation in mice with GSH/phenotype readouts; Co-IP, confocal, MIA3 KO with GSH/proliferation assays","pmids":["37014882","37948019"],"confidence":"Medium","gaps":["GSH-independent contribution to cachexia not identified","MIA3-CHAC1 functional mechanism from single lab"]},{"year":2024,"claim":"Uncovered a metabolic moonlighting function and additional post-transcriptional control, with CHAC1 bridging UBA2-PKM2 SUMOylation and m6A/YTHDF2 destabilizing CHAC1 mRNA.","evidence":"MS proteomics, Co-IP, SUMOylation and fractionation assays, ChIP for E2F1; MeRIP/RIP, mRNA half-life, ferroptosis assays","pmids":["39368995","39667319"],"confidence":"Medium","gaps":["Whether scaffold function requires enzymatic activity unknown","Single lab for each mechanism"]},{"year":2025,"claim":"Consolidated CHAC1 as a causal in vivo mediator of ferroptotic disease and mapped an extensive regulatory network (ATF4 direct binding, BACH1, EGR1, H3K9ac/P300, NUPR1, miR-383-3p, XRN2/GDIL lncRNA).","evidence":"ChIP-seq/qPCR and Chac1 KO/haploinsufficient mice across kidney, cardiac, and other ferroptosis models; multiple promoter, Co-IP, luciferase, and RNA-stability assays","pmids":["39836526","40267214","41750596","39129295","40947783","42019644","41093083","39893168"],"confidence":"High","gaps":["Many individual regulators validated in single labs/contexts","Integration and hierarchy among regulators not established"]},{"year":null,"claim":"It remains unresolved how CHAC1's enzymatic GSH-degrading activity is mechanistically separated from its non-enzymatic moonlighting roles (Notch3 inhibition, PKM2 SUMOylation scaffolding) and whether these share or diverge in their structural determinants.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No experimental high-resolution structure of human CHAC1","Enzyme-independence of scaffold/binding functions not tested with catalytic mutants","Physiological relevance of moonlighting roles relative to GSH degradation unquantified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,2,7,9]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[13]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[13]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[13]}],"pathway":[{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[1,5,15]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[1,5,14]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[0,13]}],"complexes":[],"partners":["NOTCH3","UBA2","PKM2","MIA3","ATF4","ATF3"],"other_free_text":[]}},"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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\"method\": \"In vitro enzymatic assays, yeast overexpression with catalytic mutants, glutathione depletion measurements\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution of enzymatic activity combined with active-site mutagenesis and in vivo validation in yeast; foundational mechanistic paper replicated by subsequent studies\",\n      \"pmids\": [\"23070364\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CHAC1 is a downstream component of the ATF4-ATF3-CHOP arm of the unfolded protein response (UPR); it is not activated by the parallel XBP1 or ATF6 branches. CHAC1 overexpression enhanced apoptosis and its knockdown suppressed apoptosis (TUNEL, PARP cleavage, AIF nuclear translocation), placing it as a proapoptotic effector downstream of ATF4.\",\n      \"method\": \"siRNA knockdown of ATF4/ATF3/CHOP/XBP1/ATF6, CHAC1 overexpression plasmids, TUNEL assay, PARP cleavage, AIF nuclear translocation, expression microarray\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal loss-of-function and gain-of-function experiments with defined pathway placement; independently replicated in many subsequent studies\",\n      \"pmids\": [\"19109178\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Human CHAC1 promoter is transactivated by ATF4 and ATF3 through two cis-elements: a -267 ATF/CRE site and a novel -248 ATF/CRE modifier (ACM) element. Direct binding of ATF4 and ATF3 (and C/EBPβ) at these elements was confirmed by EMSA and ChIP. CHAC1 overexpression robustly depleted glutathione in HEK293 cells; a catalytic mutant failed to do so.\",\n      \"method\": \"Luciferase reporter with promoter deletion/mutation analysis, immunoblot-EMSA, ChIP, glutathione measurement in cells overexpressing WT vs. catalytic mutant CHAC1\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (reporter assays, EMSA, ChIP, mutagenesis, enzymatic readout) in a single rigorous study confirming both transcriptional regulation and catalytic function\",\n      \"pmids\": [\"25931127\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TRIB3 represses CHAC1 expression downstream of ATF4, protecting cells from arsenite-induced glutathione depletion and death. Two regulatory elements in the Chac1 promoter mediate induction by arsenite/ATF4 and suppression by TRIB3. Chac1 silencing in Trib3-deficient cells restored glutathione and rescued arsenite sensitivity, establishing TRIB3 as an upstream negative regulator of CHAC1.\",\n      \"method\": \"Trib3-knockout MEFs, Chac1 siRNA, promoter analysis, glutathione measurements, cell viability assays\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with KO cells plus promoter dissection and functional rescue, single lab\",\n      \"pmids\": [\"27526673\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In glioblastoma cells, TMZ-induced CHAC1 expression is controlled transcriptionally by the JNK1/c-JUN pathway. CHAC1 binds to the Notch3 protein and inhibits Notch3 activation, thereby attenuating Notch3-mediated downstream signaling; CHAC1 overexpression induced apoptosis via caspase-3/9 activation, ROS generation, increased intracellular calcium, and loss of mitochondrial membrane potential.\",\n      \"method\": \"Transcriptome microarray, CHAC1 overexpression/knockdown, co-immunoprecipitation (CHAC1–Notch3 binding), caspase activation assays, ROS measurement, mitochondrial membrane potential assay\",\n      \"journal\": \"Neuropharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP demonstrating CHAC1–Notch3 interaction supported by multiple functional readouts, single lab\",\n      \"pmids\": [\"27986595\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CHAC1 degrades glutathione in TNBC cells under cystine starvation downstream of the GCN2-eIF2α-ATF4 pathway (not PERK). CHAC1 knockdown rescued GSH levels and prevented cystine-starvation-induced necroptosis and ferroptosis, placing CHAC1 as a functional effector of GCN2-driven cell death.\",\n      \"method\": \"CHAC1 siRNA knockdown, GSH measurement, cell death assays with pathway inhibitors (Nec-1, MLKL inhibitor, deferoxamine, ferrostatin-1), GCN2/PERK pathway analysis\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic loss-of-function with defined biochemical readout (GSH) and pharmacological dissection of cell death pathway, single lab\",\n      \"pmids\": [\"29383104\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"cagA-positive H. pylori infection triggers CHAC1 overexpression in gastric epithelial cells, leading to glutathione degradation, ROS accumulation, and TP53 somatic mutations. A catalytically inactive CHAC1 mutant did not induce TP53 mutations, confirming that CHAC1's glutathione-degrading enzymatic activity is necessary for ROS-mediated genotoxicity.\",\n      \"method\": \"CHAC1 overexpression (WT vs. catalytic mutant), CHAC1 siRNA, TP53 mutation sequencing, glutathione and ROS measurement in AGS cells\",\n      \"journal\": \"FEBS open bio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — active-site mutagenesis linking catalytic activity to downstream genotoxic phenotype, single lab\",\n      \"pmids\": [\"29632819\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Zebrafish Chac1 is a glutathione-degrading enzyme (acting only on reduced GSH) that is required for calcium signaling during embryonic development. chac1 morphants showed attenuated calcium transients (measured by GCaMP6s) and developmental defects in myotome, brain, and heart; these phenotypes were rescued by WT Chac1 but not catalytically inactive Chac1. Intracellular glutathione redox potential (monitored by Grx1-roGFP2) is an upstream activator of calcium signaling.\",\n      \"method\": \"Zebrafish morpholino knockdown, rescue with WT and catalytically inactive Chac1, live calcium imaging (GCaMP6s), glutathione redox sensor (Grx1-roGFP2)\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vivo reconstitution with catalytic mutant rescue, orthogonal live reporters for both redox and calcium, mechanistically rigorous\",\n      \"pmids\": [\"31189567\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CHAC1 transcription in humans and mice is induced by ATF4 through synergistic ATF/CRE and CARE (amino acid response element) sequences in the CHAC1 promoter. CHOP (DDIT3) exerts a novel inhibitory effect on CHAC1 transcription via the ATF/CRE motif through an indirect mechanism; direct binding of CHOP to the ATF/CRE was not detected by EMSA.\",\n      \"method\": \"Promoter-luciferase reporter assays, deletion/mutation analysis, IM-EMSA for ATF4 and CHOP binding, ATF4 overexpression in human and mouse cells\",\n      \"journal\": \"Biochemistry and biophysics reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple promoter dissection methods, but CHOP inhibitory mechanism is indirect and not fully resolved; single lab\",\n      \"pmids\": [\"33102815\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Active-site residues of human ChaC1 forming direct interactions with glutathione were identified: Y38, G39, S40, L41 (38-YGSL-41), D68, R72, E115, and Y143. Residues exclusive to the ChaC family (forming the active-site surface) are required for structural stability. Validation was performed by in silico docking, MD simulations, MMGBSA binding energy, and in vivo yeast activity assays with point mutants.\",\n      \"method\": \"Molecular docking of glutathione with modeled hChaC1, MD simulations, MMGBSA binding energy calculations, yeast in vivo activity assays of site-directed mutants\",\n      \"journal\": \"Proteins\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — combined computational and experimental (yeast in vivo) validation of active-site residues; in vitro enzymatic reconstitution not directly reported in abstract\",\n      \"pmids\": [\"36456186\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"DJ-1 (PARK7) binds the basic leucine zipper domain of ATF3 (shown by bimolecular fluorescence complementation) and inhibits ATF3 binding to the CHAC1 promoter, thereby downregulating CHAC1 expression and reducing glutathione degradation in astrocytes. DJ-1 knockout significantly upregulated CHAC1.\",\n      \"method\": \"DJ-1 knockout, high-throughput sequencing, bimolecular fluorescence complementation (BiFC) for DJ-1–ATF3 interaction, promoter-binding assay, GSH measurement\",\n      \"journal\": \"Neuroscience research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — BiFC protein interaction plus KO phenotype linked to CHAC1 expression and GSH levels; single lab\",\n      \"pmids\": [\"35988816\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"MIA3 binds directly to CHAC1 protein (co-immunoprecipitation and confocal co-localization) and promotes CHAC1 expression and glutathione degradation, thereby promoting hepatocellular carcinoma cell growth and metastasis. MIA3 knockout reduced CHAC1 expression and slowed GSH degradation.\",\n      \"method\": \"Co-immunoprecipitation, confocal microscopy, MIA3 overexpression/knockout, RNA-seq, GSH measurement, cell proliferation/migration/invasion assays\",\n      \"journal\": \"Molecular and cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — reciprocal Co-IP and confocal co-localization establishing protein interaction, linked to enzymatic/functional readout; single lab\",\n      \"pmids\": [\"37948019\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CHAC1 inactivation via CRISPR/Cas9 knock-in of an enzyme-inactivating mutation preserved muscle glutathione levels under fasting, cancer cachexia, and chemotherapy conditions, but failed to prevent muscle wasting, demonstrating that glutathione preservation alone is insufficient to protect against cachexia-induced muscle loss.\",\n      \"method\": \"CRISPR/Cas9 knock-in of enzymatic-inactivating mutation in mice, glutathione measurement, muscle wasting phenotypic analysis under multiple cachexia models\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vivo catalytic knock-in with direct biochemical (GSH) and phenotypic readout; single lab, but rigorous genetic approach\",\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, activating glycolysis-related gene expression and the Warburg effect in lung adenocarcinoma cells. E2F Transcription Factor 1 was identified as a transcriptional activator of CHAC1 by directly binding the CHAC1 promoter.\",\n      \"method\": \"Shotgun mass spectrometry-based proteomics, RNA-seq, co-IP for UBA2-CHAC1-PKM2 complex, SUMOylation assays, nuclear/cytoplasmic fractionation, ChIP for E2F1 at CHAC1 promoter, CHAC1 overexpression/knockdown in vitro and in vivo\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mass spectrometry-identified complex confirmed by Co-IP, with functional SUMOylation and localization readouts; single lab\",\n      \"pmids\": [\"39368995\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ATF4 directly binds the CHAC1 promoter (ChIP-seq and ChIP-qPCR) in renal tubular cells; ATF4 depletion inhibited CHAC1 upregulation and pro-ferroptotic responses induced by oxalate, establishing ATF4 as the direct transcriptional activator of CHAC1 in the ER stress-dependent ferroptosis pathway in calcium oxalate kidney stone models.\",\n      \"method\": \"ChIP-seq, ChIP-qPCR, ATF4 siRNA knockdown, 4-PBA (ER stress inhibitor) treatment, CHAC1 knockout, GSH/lipid peroxidation/Fe2+ measurements in vivo and in vitro\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — ChIP-seq/ChIP-qPCR directly demonstrating ATF4 occupancy at CHAC1 promoter, combined with in vivo genetic KO and biochemical ferroptosis readouts\",\n      \"pmids\": [\"39836526\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CHAC1 haploinsufficiency (Chac1+/− mice) conferred resilience to kidney disease (folic acid nephropathy, adenine-induced CKD, diabetic nephropathy). Tubule cells from Chac1+/− mice showed increased glutathione, decreased lipid peroxidation, improved viability, and protection against ferroptosis, establishing CHAC1 as a mediator of kidney disease through glutathione degradation and ferroptosis.\",\n      \"method\": \"CRISPR-generated Chac1+/− mice, multiple kidney disease models, primary tubule cell isolation, GSH measurement, lipid peroxidation assays, ferroptosis gene expression analysis, correlation with kidney biopsy protein data\",\n      \"journal\": \"Science translational medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vivo genetic loss-of-function in multiple disease models with direct biochemical readouts (GSH, lipid peroxidation, ferroptosis markers); replicated across multiple model systems\",\n      \"pmids\": [\"40267214\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The transcription factor BACH1 activates the CHAC1 promoter (dual-luciferase assay). BACH1 silencing attenuated ferroptosis by suppressing CHAC1 and restoring the GSH-GPX4 axis in cardiomyocytes. CHAC1 overexpression depleted GSH, suppressed GPX4, and enhanced lipid peroxidation during myocardial ischemia-reperfusion, while CHAC1 knockdown was partially protective; in vivo AAV-mediated CHAC1 overexpression worsened cardiac dysfunction and enlarged infarct area.\",\n      \"method\": \"Dual-luciferase promoter assay for BACH1-CHAC1, BACH1 siRNA, CHAC1 overexpression/knockdown, AAV-mediated cardiac CHAC1 overexpression in mice, GSH/GPX4/lipid peroxidation assays\",\n      \"journal\": \"Antioxidants\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — promoter assay plus in vivo AAV and genetic KD with multiple biochemical readouts; single lab\",\n      \"pmids\": [\"41750596\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"GDIL lncRNA binds and relocalizes the nuclear exoribonuclease XRN2 to the cytoplasm, where GDIL serves as a scaffold for XRN2 to identify and degrade CHAC1 mRNA, thereby reducing CHAC1 protein levels, boosting GSH, and promoting platinum resistance in colorectal cancer.\",\n      \"method\": \"RNA pulldown, GDIL-XRN2 interaction assays, metabolomic and metabolic flux analysis, CHAC1 mRNA stability assays, antisense oligonucleotide suppression in cell lines and patient-derived xenografts\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods identifying post-transcriptional regulation of CHAC1 mRNA by XRN2 scaffold; single lab\",\n      \"pmids\": [\"39893168\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"m6A methylation by METTL3/14 destabilizes CHAC1 mRNA via YTHDF2-mediated decay (RIP assays confirmed METTL3/YTHDF2-CHAC1 mRNA interaction). Arsenic treatment reduced METTL3/14 expression, inhibited m6A modification of CHAC1 mRNA, and increased CHAC1 mRNA half-life (~2-fold), leading to elevated CHAC1 protein, glutathione degradation, GPX4 suppression, and β-cell ferroptosis. METTL3 overexpression restored CHAC1 mRNA degradation and alleviated arsenic-induced dysfunction.\",\n      \"method\": \"MeRIP (m6A sequencing), RIP assay, METTL3/14 overexpression, CHAC1 mRNA half-life measurement (actinomycin D), CHAC1 knockdown, GSH/GPX4/ferroptosis assays\",\n      \"journal\": \"Ecotoxicology and environmental safety\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MeRIP and RIP demonstrating m6A-YTHDF2 regulation of CHAC1 mRNA stability, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"39667319\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NUPR1 physically interacts with ATF4 (co-immunoprecipitation in HK2 cells) and suppresses ferroptosis in renal ischemia-reperfusion injury by inhibiting the ATF4-CHAC1 pathway; NUPR1 overexpression reduced CHAC1 expression while NUPR1 knockdown enhanced ferroptosis via ATF4-CHAC1 activation.\",\n      \"method\": \"Co-immunoprecipitation (NUPR1–ATF4), NUPR1 overexpression/knockdown, renal IRI mouse model, hypoxia-reoxygenation HK2 cell model, ferroptosis marker measurement\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP establishing NUPR1-ATF4 protein interaction plus in vivo and in vitro functional consequences for CHAC1-dependent ferroptosis; single lab\",\n      \"pmids\": [\"42019644\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"H3K9 acetylation at the CHAC1 promoter (mediated by increased histone acetyltransferase P300) is required for DHA-induced transcriptional upregulation of CHAC1 in hepatic stellate cells. ATF4 binds two essential CHAC1 promoter regions (-212 to -199 bp and -269 to -257 bp), confirmed by luciferase reporter assays and ChIP-qPCR; inhibiting histone acetylation or ATF4 both blocked CHAC1 induction and reduced ferroptosis.\",\n      \"method\": \"ChIP-qPCR for H3K9ac and ATF4, luciferase reporter with WT and mutated CHAC1 promoter, P300 inhibition, ATF4 knockdown, RNA-seq, in vivo CCl4 fibrosis model\",\n      \"journal\": \"Chinese medical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — ChIP, reporter assays, and in vivo experiments establishing epigenetic (H3K9ac/P300) and ATF4 regulation of CHAC1 transcription; single lab\",\n      \"pmids\": [\"40947783\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"EGR1 transcriptionally activates CHAC1 expression; EGR1 binding to the CHAC1 promoter was validated by chromatin immunoprecipitation and dual-luciferase reporter assays. EGR1-driven CHAC1 upregulation promotes ferroptosis in keratinocytes; caffeic acid inhibits ferroptosis by suppressing the EGR1-CHAC1 axis.\",\n      \"method\": \"ChIP assay, dual-luciferase reporter assay, EGR1/CHAC1 overexpression and knockdown plasmids, ferroptosis markers (MDA, ROS, Fe2+, GSH), flow cytometry\",\n      \"journal\": \"Current molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP and reporter assays directly demonstrating EGR1 binding and transactivation of CHAC1 promoter; single lab\",\n      \"pmids\": [\"39129295\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Juglone (a naturally occurring naphthoquinone) inhibits ChaC1 enzymatic activity with an IC50 of 8.7 µM in vitro; inhibition is non-competitive with glutathione and occurs through a novel mechanism independent of cysteine adduct formation (a cysteine-free ChaC1 variant was still inhibited). Plumbagin was also identified as an effective inhibitor.\",\n      \"method\": \"Yeast-based high-throughput functional screens, in vitro ChaC1 enzymatic assays, kinetic studies (competitive vs. non-competitive), cysteine-free ChaC1 variant testing\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic characterization of inhibitor mechanism with genetic variant controls; single lab, peer-reviewed publication\",\n      \"pmids\": [\"39400295\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CHAC1 promotes ferroptosis in sepsis-exposed cardiomyocytes via the ATF4-CHOP-CHAC1 axis. miR-383-3p directly targets ATF4 mRNA (validated by dual-luciferase assay), thereby suppressing the ATF4-CHOP-CHAC1 pathway; ATF4 overexpression abolished miR-383-3p's protective effects, and ATF4 knockdown phenocopied miR-383-3p suppression of CHAC1 and ferroptosis.\",\n      \"method\": \"Dual-luciferase reporter assay (miR-383-3p–ATF4), ATF4 overexpression/knockdown, LPS cardiomyocyte model, miR-383-3p mimics/inhibitors, in vivo sepsis mouse model, ferroptosis markers\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — luciferase validation of miRNA-ATF4 interaction combined with genetic epistasis placing ATF4 upstream of CHOP-CHAC1; single lab\",\n      \"pmids\": [\"41093083\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CHAC1 is a cytosolic γ-glutamyl cyclotransferase that specifically degrades glutathione (not other γ-glutamyl peptides), depleting intracellular GSH and thereby promoting oxidative stress, ferroptosis, and apoptosis; its transcription is activated primarily by ATF4 (and ATF3) through defined ATF/CRE and CARE promoter elements downstream of the PERK-eIF2α arm of the UPR, and is further modulated by additional transcription factors (EGR1, BACH1), epigenetic marks (H3K9 acetylation via P300), m6A mRNA modification (METTL3/YTHDF2), and regulatory proteins (TRIB3 as repressor, NUPR1 via ATF4 interaction, DJ-1 via ATF3 binding); beyond GSH degradation, CHAC1 also acts as a scaffold enhancing PKM2 SUMOylation to drive nuclear translocation and the Warburg effect, binds Notch3 to inhibit its signaling, and is required for calcium transient generation during zebrafish development through its ability to alter intracellular redox potential.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CHAC1 is a cytosolic γ-glutamyl cyclotransferase that specifically degrades intracellular reduced glutathione, and through this enzymatic depletion of GSH it drives oxidative stress, ferroptosis, and apoptosis across diverse cell types [#0, #7, #15]. Its substrate specificity is narrow — it acts on glutathione but not other γ-glutamyl peptides — and catalysis depends on a defined active-site surface in which residues such as Y38–L41, D68, R72, E115, and Y143 contact glutathione; catalytically dead mutants fail to deplete GSH or produce downstream phenotypes [#0, #9]. CHAC1 functions principally as a downstream effector of the integrated stress response: it is a proapoptotic component of the ATF4–ATF3–CHOP arm of the UPR, with ATF4 (and ATF3) directly transactivating the CHAC1 promoter through ATF/CRE and CARE elements [#1, #2, #14], and it operates downstream of both PERK and GCN2 branches depending on the stress, mediating cystine-starvation necroptosis and ferroptosis [#5]. In vivo genetic studies establish that CHAC1's GSH-degrading activity is required for pathology: Chac1 haploinsufficiency or catalytic inactivation preserves glutathione, suppresses lipid peroxidation, and confers resistance to ferroptosis-driven kidney disease [#14, #15], although GSH preservation alone is insufficient to prevent cachexia-induced muscle wasting [#12]. Beyond GSH degradation, CHAC1 binds Notch3 and inhibits its activation [#4], bridges UBA2 and PKM2 to enhance PKM2 SUMOylation and nuclear translocation driving the Warburg effect [#13], and is required for glutathione-redox-dependent calcium transients during zebrafish development [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"Established CHAC1 as a proapoptotic effector positioned specifically within the ATF4-ATF3-CHOP arm of the UPR, distinguishing it from the parallel XBP1/ATF6 branches.\",\n      \"evidence\": \"siRNA knockdown of UPR transcription factors plus CHAC1 gain/loss-of-function with apoptosis readouts (TUNEL, PARP cleavage, AIF translocation)\",\n      \"pmids\": [\"19109178\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular activity of CHAC1 not yet defined at this stage\", \"Direct promoter binding not yet demonstrated\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Defined the molecular function of CHAC1 as a γ-glutamyl cyclotransferase that specifically degrades glutathione, and showed this enzymatic activity is required for its proapoptotic effect.\",\n      \"evidence\": \"In vitro enzymatic assays and yeast overexpression with catalytic E>Q mutants, glutathione depletion measurements\",\n      \"pmids\": [\"23070364\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Active-site architecture not resolved\", \"Mammalian in vivo relevance not yet established\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identified the cis-regulatory basis of CHAC1 induction, mapping direct ATF4/ATF3 binding to ATF/CRE and ACM promoter elements and confirming enzymatic GSH depletion in human cells.\",\n      \"evidence\": \"Promoter deletion luciferase reporters, EMSA, ChIP, and WT-vs-catalytic-mutant GSH measurement in HEK293\",\n      \"pmids\": [\"25931127\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cell-type-specific cofactors not addressed\", \"Quantitative contribution of each element in vivo unknown\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Revealed both negative transcriptional control and a non-enzymatic moonlighting function, with TRIB3 repressing CHAC1 and CHAC1 binding Notch3 to inhibit its signaling.\",\n      \"evidence\": \"Trib3-KO MEFs with promoter analysis and rescue; transcriptome profiling, Co-IP, and apoptosis/ROS/calcium/mitochondrial assays in glioblastoma cells\",\n      \"pmids\": [\"27526673\", \"27986595\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"CHAC1-Notch3 interaction from single Co-IP without reciprocal/structural validation\", \"Whether Notch3 inhibition is enzyme-dependent unresolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Extended CHAC1's effector role to a second stress branch, showing it executes GCN2-eIF2α-ATF4-driven necroptosis and ferroptosis under cystine starvation, independent of PERK.\",\n      \"evidence\": \"CHAC1 siRNA with GSH measurement and pharmacological cell-death dissection (Nec-1, ferrostatin-1, deferoxamine) in TNBC cells\",\n      \"pmids\": [\"29383104\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Branch selectivity between PERK and GCN2 not mechanistically explained\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Demonstrated that CHAC1 enzymatic GSH degradation has genotoxic consequences, linking H. pylori-induced CHAC1 to ROS accumulation and TP53 mutation.\",\n      \"evidence\": \"WT-vs-catalytic-mutant overexpression, siRNA, TP53 sequencing, GSH/ROS measurement in AGS gastric cells\",\n      \"pmids\": [\"29632819\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct link between specific ROS species and mutation spectrum not established\", \"Single lab\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Established a developmental and physiological role beyond cell death, showing GSH-redox-dependent CHAC1 activity is required for calcium transient generation in vivo.\",\n      \"evidence\": \"Zebrafish morpholino knockdown with WT vs catalytically-inactive rescue, GCaMP6s calcium imaging, Grx1-roGFP2 redox sensing\",\n      \"pmids\": [\"31189567\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular link between redox potential and calcium machinery not defined\", \"Mammalian conservation of this role untested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Refined transcriptional logic by showing ATF4 acts through synergistic ATF/CRE and CARE elements while CHOP exerts an indirect inhibitory effect, complicating the simple ATF4-CHOP-CHAC1 chain.\",\n      \"evidence\": \"Promoter-luciferase, deletion/mutation analysis, IM-EMSA for ATF4 and CHOP in human and mouse cells\",\n      \"pmids\": [\"33102815\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of indirect CHOP repression unresolved (no direct binding detected)\", \"Single lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Provided structural insight into substrate recognition and uncovered additional transcriptional repression via DJ-1-ATF3 interaction.\",\n      \"evidence\": \"Molecular docking/MD/MMGBSA with yeast point-mutant validation of active-site residues; BiFC of DJ-1-ATF3 with promoter-binding and GSH assays\",\n      \"pmids\": [\"36456186\", \"35988816\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No experimental crystal/cryo-EM structure\", \"In vitro enzymatic reconstitution of active-site mutants not directly reported\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Separated CHAC1's enzymatic and disease functions and added a direct protein partner, showing catalytic inactivation preserves muscle GSH yet fails to prevent cachexia, while MIA3 binds and stabilizes CHAC1.\",\n      \"evidence\": \"CRISPR knock-in of enzyme-inactivating mutation in mice with GSH/phenotype readouts; Co-IP, confocal, MIA3 KO with GSH/proliferation assays\",\n      \"pmids\": [\"37014882\", \"37948019\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"GSH-independent contribution to cachexia not identified\", \"MIA3-CHAC1 functional mechanism from single lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Uncovered a metabolic moonlighting function and additional post-transcriptional control, with CHAC1 bridging UBA2-PKM2 SUMOylation and m6A/YTHDF2 destabilizing CHAC1 mRNA.\",\n      \"evidence\": \"MS proteomics, Co-IP, SUMOylation and fractionation assays, ChIP for E2F1; MeRIP/RIP, mRNA half-life, ferroptosis assays\",\n      \"pmids\": [\"39368995\", \"39667319\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether scaffold function requires enzymatic activity unknown\", \"Single lab for each mechanism\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Consolidated CHAC1 as a causal in vivo mediator of ferroptotic disease and mapped an extensive regulatory network (ATF4 direct binding, BACH1, EGR1, H3K9ac/P300, NUPR1, miR-383-3p, XRN2/GDIL lncRNA).\",\n      \"evidence\": \"ChIP-seq/qPCR and Chac1 KO/haploinsufficient mice across kidney, cardiac, and other ferroptosis models; multiple promoter, Co-IP, luciferase, and RNA-stability assays\",\n      \"pmids\": [\"39836526\", \"40267214\", \"41750596\", \"39129295\", \"40947783\", \"42019644\", \"41093083\", \"39893168\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Many individual regulators validated in single labs/contexts\", \"Integration and hierarchy among regulators not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how CHAC1's enzymatic GSH-degrading activity is mechanistically separated from its non-enzymatic moonlighting roles (Notch3 inhibition, PKM2 SUMOylation scaffolding) and whether these share or diverge in their structural determinants.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No experimental high-resolution structure of human CHAC1\", \"Enzyme-independence of scaffold/binding functions not tested with catalytic mutants\", \"Physiological relevance of moonlighting roles relative to GSH degradation unquantified\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 2, 7, 9]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [13]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [13]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [13]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [1, 5, 15]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [1, 5, 14]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [0, 13]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"Notch3\", \"UBA2\", \"PKM2\", \"MIA3\", \"ATF4\", \"ATF3\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}