{"gene":"CBS","run_date":"2026-06-09T22:57:17","timeline":{"discoveries":[{"year":2004,"finding":"Tandem pairs of CBS domains from cystathionine beta-synthase bind AMP, ATP, or S-adenosyl methionine; disease-causing mutations in the CBS domains impair this nucleotide binding, establishing CBS domains as adenosine-derivative sensing modules.","method":"In vitro binding assays with purified CBS domain proteins; functional assessment of disease mutants","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct biochemical binding assay with multiple proteins and disease mutant validation, replicated across multiple CBS-domain-containing proteins","pmids":["14722619"],"is_preprint":false},{"year":2013,"finding":"Human CBS contains two classes of SAM-binding sites in the C-terminal regulatory domain: a high-affinity set of two sites involved in kinetic stabilization, and a low-affinity set of four sites responsible for enzyme activation. SAM-induced stabilization modulates steady-state CBS levels in vivo.","method":"Calorimetric methods (ITC), functional enzyme assays, kinetic modeling on purified WT and homocystinuria mutant CBS proteins","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — multiple orthogonal methods (calorimetry + functional assays + kinetic modeling) in single lab on purified protein","pmids":["22985361"],"is_preprint":false},{"year":2010,"finding":"Eight homocystinuria-causing CBS missense mutants (P49L, P78R, A114V, R125Q, E176K, P422L, I435T, S466L) can be rescued by chemical chaperones during expression, yielding tetrameric enzymes with restored catalytic activity; R125Q and E176K fail to respond to SAM stimulation or thermal activation, indicating their conformations cannot reach the activated state.","method":"Purification of CBS mutant proteins expressed in E. coli with chemical chaperones; enzyme activity assays; thermal stability measurements; SAM stimulation assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstitution with purified proteins, multiple orthogonal functional readouts, single lab","pmids":["20308073"],"is_preprint":false},{"year":2012,"finding":"CBS catalyzes PLP-dependent reactions to produce cystathionine and H2S; the C. elegans CBS-1 ortholog has a unique tandem-repeat architecture with one active PLP-binding catalytic module and one inactive N-terminal module, lacks heme and the regulatory Bateman domain, forms monomers, and its knockdown causes ~10-fold elevation of homocysteine.","method":"Truncation and active-site mutagenesis of purified CBS-1; PLP-binding assays; in vivo RNAi knockdown; homocysteine measurement","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstitution with truncated/mutant proteins, multiple orthogonal methods (activity assay, mutagenesis, RNAi phenotype), single lab","pmids":["22240119"],"is_preprint":false},{"year":2018,"finding":"The p.G307S CBS mutation abolishes enzyme activity without affecting protein stability or multimerization; molecular dynamics simulation indicates the substitution restricts Y308 conformational flexibility required for formation of the pyridoxal-cystathionine catalytic intermediate.","method":"Transgenic mouse model expressing p.G307S CBS; enzyme activity assays; proteasome inhibitor treatment; molecular dynamics simulation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vivo mouse model plus in vitro biochemical characterization plus structural simulation, multiple methods in single lab","pmids":["30030379"],"is_preprint":false},{"year":2013,"finding":"Reduced SAM activation of CBS is caused by C-terminal domain mutations that produce abnormal oligomeric conformations unable to reach the activated state, while catalytic-core mutations reduce protein expression and activity; SAM-activation defect confirmed in both patient fibroblasts and recombinant mutant proteins.","method":"Retrospective fibroblast activity assays; heterologous CBS mutant expression in E. coli; Western blotting; enzyme activity in absence/presence of SAM and PLP","journal":"Journal of inherited metabolic disease","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — multiple mutant proteins characterized, but single lab, no structural validation","pmids":["23974653"],"is_preprint":false},{"year":2019,"finding":"CBS-derived H2S directly suppresses mitochondrial Complex IV activity; pharmacological inhibition of CBS (aminooxyacetate) or siRNA silencing in Down syndrome fibroblasts normalizes H2S levels, restores Complex IV activity, improves mitochondrial electron transport and ATP synthesis, and restores cell proliferation.","method":"CBS inhibition (aminooxyacetate) and siRNA knockdown in DS fibroblasts; measurement of H2S, polysulfides, mitochondrial oxygen consumption, ATP generation, Complex IV activity","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Moderate — two orthogonal loss-of-function approaches (pharmacological + siRNA) with defined cellular and biochemical phenotype, single lab","pmids":["31481613"],"is_preprint":false},{"year":2019,"finding":"Cbs overdosage is necessary and sufficient for recognition-memory deficits in Down syndrome mouse models; Cbs interacts genetically (epistasis) with Dyrk1a to control memory, and proteomic analysis implicates synaptic transmission, cell projection morphogenesis, and actin cytoskeleton pathways.","method":"Genetic deletion and transgenic overexpression of Cbs in mice; novel object recognition behavioral testing; pharmacological rescue; genetic epistasis with Dyrk1a; proteomic analysis","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 / Moderate — necessity and sufficiency established by KO and OE, genetic epistasis, pharmacological rescue, and proteomics in single lab","pmids":["30649339"],"is_preprint":false},{"year":2020,"finding":"CBS physically interacts with the core circadian protein CRY1; in cells, CBS augments CRY1-mediated repression of the CLOCK/BMAL1 complex and shortens circadian period; CRY1 binding reciprocally modulates CBS enzymatic activity; liver extracts from Cry1-/- mice show reduced CBS activity restored by exogenous WT CRY1.","method":"Co-immunoprecipitation; circadian period assays; CBS enzymatic activity in Cry1-/- liver extracts with/without exogenous CRY1; metabolomics; transgenic CbsZn/Zn mouse circadian analysis","journal":"The FEBS journal","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal functional interaction demonstrated by Co-IP, enzyme activity rescue, and in vivo mouse model with metabolomics, single lab","pmids":["32383312"],"is_preprint":false},{"year":2016,"finding":"Muscarinic receptor (M1/M3) activation in human urothelium phosphorylates CBS at Ser227 via the cGMP/PKG pathway, stimulating H2S production and cGMP, which promotes bladder relaxation; CBS (but not CSE) inhibition mimics urothelium removal and increases carbachol-induced contraction.","method":"CBS inhibition pharmacology; site-specific phosphorylation analysis (Ser227); functional bladder strip assays; T24 cell line experiments; receptor blockade","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — post-translational modification (phosphorylation) identified with pathway context and functional consequence, single lab","pmids":["27509878"],"is_preprint":false},{"year":2019,"finding":"CBS-derived H2S production is activated by NRF2 transcriptional upregulation of CBS in erastin-resistant ovarian cancer cells; CBS knockdown causes ferroptotic cell death in resistant cells, while CBS overexpression confers resistance, demonstrating NRF2/CBS signaling as a ferroptosis resistance mechanism through the transsulfuration pathway.","method":"RNAi knockdown of CBS and NRF2; CBS overexpression; cell viability and ROS measurement; metabolite analysis of transsulfuration pathway","journal":"British journal of cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — bidirectional genetic manipulation (KD and OE) with defined ferroptosis phenotype, single lab","pmids":["31819185"],"is_preprint":false},{"year":2014,"finding":"CBS regulates mitochondrial bioenergetics in ovarian cancer cells by controlling ROS production, oxygen consumption, and ATP generation; CBS silencing via nanoliposomal siRNA inhibits tumor growth and sensitizes cisplatin-resistant cells.","method":"CBS siRNA silencing (in vitro and in vivo); mitochondrial ROS, oxygen consumption, ATP measurements; orthotopic mouse model","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function in vitro and in vivo with multiple bioenergetic readouts, single lab","pmids":["24236104"],"is_preprint":false},{"year":2014,"finding":"SAM, as an allosteric CBS activator, markedly enhances CBS-mediated H2S production in vitro; low-to-intermediate SAM concentrations stimulate HCT116 colon cancer cell proliferation and bioenergetics in a CBS-dependent manner (attenuated by AOAA or CBS silencing), while high SAM concentrations inhibit proliferation by CBS-independent mechanisms.","method":"Recombinant CBS enzyme assay; HCT116 cell proliferation (xCELLigence); extracellular flux bioenergetics; CBS stable silencing; AOAA pharmacological inhibition","journal":"Nitric oxide : biology and chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro enzyme assay plus cell-based pharmacological and genetic validation, multiple orthogonal methods, single lab","pmids":["24667534"],"is_preprint":false},{"year":2015,"finding":"TLR4 activation upregulates CBS expression in dorsal root ganglion neurons via NF-κB signaling; NF-κB inhibition reduces CBS upregulation and attenuates visceral hypersensitivity in a rat IBS model.","method":"Western blot; intrathecal pharmacological inhibition of TLR4 and NF-κB; LPS stimulation of cultured DRG neurons; patch-clamp electrophysiology","journal":"World journal of gastroenterology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological and in vitro manipulation defining upstream pathway to CBS, single lab, multiple readouts","pmids":["26229403"],"is_preprint":false},{"year":2011,"finding":"Serotonin and dopamine increase CBS expression via mTOR signaling and stimulate H2S production in rat smooth muscle cells, protecting against hypothermia/rewarming-induced ROS and apoptosis; CBS inhibition (pharmacological or siRNA) abolishes these protective effects.","method":"CBS mRNA/protein measurement; siRNA knockdown; pharmacological CBS inhibition; ROS measurement; caspase activity assay; organ preservation experiments","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two orthogonal CBS loss-of-function methods with defined cytoprotective phenotype, single lab","pmids":["21829469"],"is_preprint":false},{"year":2020,"finding":"Estradiol-17β (E2) specifically stimulates CBS (but not CSE) expression and H2S production in endometrial stromal cells via estrogen receptor (blocked by ICI 182780); stromal CBS-derived H2S promotes endometrial microvascular endothelial cell angiogenesis in a paracrine manner.","method":"CBS/CSE expression analysis; ICI 182780 receptor blockade; CBS pharmacological inhibition; co-culture migration assay; in vivo endometrial analysis across hormonal states","journal":"Endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — receptor-specific regulation and functional consequence demonstrated with pharmacological inhibition, single lab","pmids":["32987401"],"is_preprint":false},{"year":2021,"finding":"H2S produced by CBS promotes S-sulfhydration of HuR (human antigen R), which decreases HuR binding to COX-2 mRNA, reducing COX-2 mRNA stability and expression; CBS heterozygous knockout mice show decreased HuR sulfhydration and increased COX-2 in colon.","method":"Biotin switch technique for S-sulfhydration; CRISPR/Cas9 CBS knockout; ActD chase assay for mRNA stability; CBS+/- mouse colitis model; immunofluorescence","journal":"Journal of advanced research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct PTM (sulfhydration) identified with functional consequence on mRNA stability, validated in vivo, multiple orthogonal methods, single lab","pmids":["36725190"],"is_preprint":false},{"year":2021,"finding":"CBS-derived H2S promotes testosterone synthesis in MLTC-1 cells by sulfhydrylating PDE4A and PDE8A (reducing their activity), thereby elevating cAMP and activating PKA pathway; CBS overexpression increases StAR, P450scc, P450c17 and 3β-HSD expression.","method":"CBS overexpression/knockdown; CBS/H2S measurement; biotin switch technique for PDE sulfhydrylation; PKA pathway analysis; RT-qPCR and western blot","journal":"Journal of cellular and molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct PTM (sulfhydration) of substrate PDE identified by biotin switch, functional pathway consequence demonstrated, single lab","pmids":["33713531"],"is_preprint":false},{"year":2021,"finding":"CBS knockout using CRISPR in normal gastric epithelial cells induces widespread DNA methylation changes overlapping with gastric cancer CIMP patterns, and CBS loss reduces H2S and increases NF-κB activity, linking CBS to both epigenetic regulation and inflammation.","method":"CRISPR deletion of CBS; global DNA methylation profiling; NF-κB activity assay; H2S measurement; proteomics; comparison with primary gastric cancer CIMP patterns","journal":"Genome biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR KO with defined molecular phenotypes (methylation, NF-κB), single lab","pmids":["34074348"],"is_preprint":false},{"year":2023,"finding":"ATF6 directly binds the CBS gene promoter and transcriptionally upregulates CBS expression; ATF6-driven CBS activity increases H2S synthesis, which sulfhydrates SIRT1, reducing lipogenesis and inflammation in fatty liver.","method":"ATF6 liver-specific knockout mice; chromatin immunoprecipitation (ChIP) at CBS promoter; H2S measurement; SIRT1 sulfhydration assay; high-fat diet model","journal":"European journal of medical research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP establishes direct ATF6 binding at CBS promoter with downstream H2S/SIRT1 consequence, single lab","pmids":["38007457"],"is_preprint":false},{"year":2024,"finding":"ATF3 positively regulates CBS expression under cystine deprivation, conferring ferroptosis resistance in colorectal cancer; CBS suppression sensitizes cells to ferroptosis by targeting the mitochondrial TCA cycle; ATF3-CBS axis positively correlates with CRC progression.","method":"ATF3 and CBS gain/loss-of-function; ferroptosis assays; metabolic (TCA cycle) analysis; CRC patient correlation","journal":"Redox biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — bidirectional genetic manipulation of ATF3-CBS axis with defined ferroptosis and metabolic readouts, single lab","pmids":["38490069"],"is_preprint":false},{"year":2019,"finding":"CBS deficiency suppresses erythropoiesis by reducing expression of heme biosynthetic enzymes ALAS2 and FECH, heme exporter FLVCR, HIF-2α, EPO, and EPOR in CBS knockout mice, while increasing IL-6, hepcidin, and iron content.","method":"CBS-/-, CBS+/-, and CBS+/+ mouse comparison; Western blot and qPCR for heme pathway proteins; blood and bone marrow iron content measurement","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with defined molecular phenotype across multiple pathway components, single lab","pmids":["31551410"],"is_preprint":false},{"year":2003,"finding":"CBS protein is localized predominantly in the cytosol of neurons, with strongest expression in Purkinje cell bodies/neuronal processes and Ammon's horn neurons in adult mouse brain; CBS expression peaks during cerebellar development and is present in liver from early embryonic stages.","method":"In situ hybridization; Northern blotting; immunohistochemistry in mouse brain and developmental tissues","journal":"The journal of histochemistry and cytochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct subcellular/tissue localization by immunohistochemistry and in situ hybridization, single lab, no functional consequence linked","pmids":["12588964"],"is_preprint":false},{"year":2019,"finding":"CBS localizes to both cytosol and mitochondria in Down syndrome fibroblasts, and mitochondrial CBS-derived H2S is responsible for tonic suppression of Complex IV.","method":"CBS subcellular fractionation and localization in DS fibroblasts; CBS inhibition/siRNA with Complex IV activity measurement","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct fractionation establishing mitochondrial localization with functional consequence on Complex IV, single lab","pmids":["31481613"],"is_preprint":false},{"year":2001,"finding":"A 31 bp VNTR spanning the exon 13-intron 13 boundary of the CBS gene is associated with reduced CBS enzyme activity in patient fibroblasts (measured directly) and elevated post-methionine load homocysteine; RT-PCR shows evidence of alternative splicing at this junction dependent on repeat number.","method":"CBS enzyme activity in cultured fibroblasts; RT-PCR; VNTR genotyping in patients and controls","journal":"European journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct enzyme activity measurement in primary fibroblasts correlating with genotype, with RT-PCR evidence of splicing mechanism, single lab","pmids":["11528503"],"is_preprint":false},{"year":2012,"finding":"CBS (alongside CSE) is expressed in adrenocortical cells and is required for H2S generation; CBS/CSE inhibition or siRNA knockdown causes mitochondrial oxidative stress and dysfunction and blunts corticosterone responses to ACTH; LPS inhibits CBS/CSE expression and H2S production in adrenal glands.","method":"CBS/CSE inhibitors and siRNA in adrenocortical cells; in vivo adrenal suppression model; mitochondrial function assays; corticosterone measurement","journal":"Antioxidants & redox signaling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two orthogonal loss-of-function approaches in vitro and in vivo with defined steroidogenic phenotype, single lab","pmids":["24702258"],"is_preprint":false}],"current_model":"CBS (cystathionine β-synthase) is a PLP- and heme-containing tetrameric enzyme that catalyzes the first committed step of the transsulfuration pathway (condensation of serine and homocysteine to cystathionine) and produces H2S; its activity is allosterically activated by SAM binding to two classes of sites in the C-terminal regulatory (Bateman/CBS) domain, it is post-translationally regulated by cGMP/PKG-dependent phosphorylation at Ser227 and by interaction with the circadian protein CRY1, it localizes to both cytosol and mitochondria where CBS-derived H2S tonically suppresses Complex IV, it acts as a transcriptional target of NRF2, ATF3, ATF6, and NF-κB, and it promotes ferroptosis resistance and cancer cell bioenergetics through H2S-mediated sulfhydration of downstream targets including HuR and phosphodiesterases."},"narrative":{"mechanistic_narrative":"CBS (cystathionine β-synthase) is a PLP-dependent enzyme that catalyzes the condensation of serine and homocysteine to cystathionine and concomitantly generates hydrogen sulfide (H2S), positioning it as the entry point of the transsulfuration pathway and a major source of signaling sulfide [PMID:22240119]. Enzyme output is set by allosteric control through the C-terminal regulatory (Bateman/CBS) domain, which binds adenosine derivatives including AMP, ATP, and S-adenosylmethionine; SAM occupies two classes of sites — high-affinity sites that kinetically stabilize the protein and modulate its steady-state level, and low-affinity sites that drive catalytic activation [PMID:14722619, PMID:22985361, PMID:24667534]. Homocystinuria-causing missense mutations act by distinct routes: catalytic-core substitutions reduce activity or expression (and one, G307S, restricts the conformational flexibility needed to form the catalytic intermediate), whereas C-terminal mutations trap abnormal oligomers that cannot reach the SAM-activated state, defects partially correctable by chemical chaperones [PMID:20308073, PMID:30030379, PMID:23974653]. Beyond intrinsic regulation, CBS is post-translationally controlled by cGMP/PKG-dependent phosphorylation at Ser227 and by physical interaction with the circadian repressor CRY1, which reciprocally modulates CBS activity while CBS augments CRY1-mediated repression of CLOCK/BMAL1 [PMID:32383312, PMID:27509878]. CBS is a transcriptional target of multiple stress and inflammatory programs — NRF2, ATF3, ATF6, and NF-κB — that converge to raise CBS-derived H2S [PMID:31819185, PMID:26229403, PMID:38007457, PMID:38490069]. Downstream, this H2S acts through protein S-sulfhydration of targets including HuR (destabilizing COX-2 mRNA), phosphodiesterases PDE4A/PDE8A (elevating cAMP/PKA signaling for steroidogenesis), and SIRT1 (limiting hepatic lipogenesis), and it tonically suppresses mitochondrial Complex IV from a mitochondrial pool of the enzyme [PMID:36725190, PMID:33713531, PMID:38007457, PMID:31481613]. Functionally, CBS-driven sulfide supports cancer cell bioenergetics and ferroptosis resistance, modulates erythropoiesis and the heme pathway, and contributes to cytoprotection and angiogenesis across tissues [PMID:31819185, PMID:24236104, PMID:38490069, PMID:31551410].","teleology":[{"year":2001,"claim":"Established a genetic determinant of CBS activity by linking a regulatory-region VNTR to reduced enzyme function, connecting sequence variation to the transsulfuration phenotype.","evidence":"CBS enzyme activity in patient fibroblasts, RT-PCR splicing analysis, and VNTR genotyping","pmids":["11528503"],"confidence":"Medium","gaps":["Mechanism linking repeat number to splicing outcome not fully resolved","Effect size on clinical phenotype unclear"]},{"year":2003,"claim":"Defined the tissue and subcellular distribution of CBS, showing predominantly cytosolic neuronal expression with developmental regulation, framing where the enzyme acts.","evidence":"In situ hybridization, Northern blot, and immunohistochemistry in mouse brain and embryonic tissues","pmids":["12588964"],"confidence":"Medium","gaps":["No functional consequence linked to localization","Mitochondrial pool not yet identified"]},{"year":2004,"claim":"Identified the CBS domains as adenosine-derivative sensing modules, explaining how nucleotide ligands and disease mutations converge on the enzyme's regulatory region.","evidence":"In vitro binding assays with purified CBS-domain proteins and disease-mutant functional tests","pmids":["14722619"],"confidence":"High","gaps":["Quantitative coupling of ligand binding to catalytic rate not defined","Structural basis of mutant binding loss not resolved here"]},{"year":2013,"claim":"Resolved the dual logic of SAM regulation — distinguishing high-affinity stabilizing sites that set protein level from low-affinity activating sites — explaining how a single metabolite both stabilizes and activates CBS.","evidence":"ITC, functional enzyme assays, and kinetic modeling on purified WT and homocystinuria-mutant CBS","pmids":["22985361","23974653"],"confidence":"High","gaps":["Structural geometry of the six sites not directly visualized","In vivo contribution of stabilization vs activation hard to disentangle"]},{"year":2012,"claim":"Dissected the conserved catalytic core through a divergent ortholog lacking heme and the Bateman domain, isolating the PLP-dependent module responsible for cystathionine/H2S production and homocysteine homeostasis.","evidence":"Truncation/active-site mutagenesis of C. elegans CBS-1, PLP-binding assays, and RNAi knockdown with homocysteine measurement","pmids":["22240119"],"confidence":"High","gaps":["Ortholog lacks the regulatory features of human CBS","Heme contribution to catalysis not addressed"]},{"year":2010,"claim":"Classified homocystinuria mutations mechanistically and showed chemical chaperones can rescue folding-defective mutants, separating conformational defects from irreversibly inactivated states.","evidence":"Purified CBS mutants expressed with chemical chaperones, activity, thermal stability, and SAM-stimulation assays","pmids":["20308073"],"confidence":"High","gaps":["In vivo chaperone efficacy not tested here","R125Q/E176K activation failure structurally unexplained"]},{"year":2018,"claim":"Pinpointed a single-residue catalytic mechanism by which G307S abolishes activity without destabilizing the protein, isolating conformational flexibility at the active site as essential for intermediate formation.","evidence":"Transgenic G307S mouse, enzyme assays, proteasome inhibition, and molecular dynamics simulation","pmids":["30030379"],"confidence":"High","gaps":["Simulation prediction not confirmed by crystallography","Generalizability to other core mutations untested"]},{"year":2011,"claim":"Linked extracellular signals to CBS-dependent cytoprotection, showing monoamine/mTOR-driven CBS expression generates H2S that limits oxidative apoptosis.","evidence":"CBS mRNA/protein measurement, siRNA and pharmacological inhibition, ROS and caspase assays in smooth muscle cells","pmids":["21829469"],"confidence":"Medium","gaps":["Direct mTOR-to-CBS transcriptional link not mapped","H2S effector targets not identified"]},{"year":2014,"claim":"Connected CBS-derived H2S to cancer bioenergetics, demonstrating that SAM-activated CBS fuels tumor proliferation, mitochondrial respiration, and chemoresistance.","evidence":"Recombinant enzyme assays, CBS silencing in vitro/in vivo, bioenergetic flux measurements, and orthotopic tumor model","pmids":["24667534","24236104"],"confidence":"Medium","gaps":["Biphasic SAM effect mechanism (CBS-independent at high dose) unresolved","Direct sulfhydration targets in this context not identified"]},{"year":2016,"claim":"Identified Ser227 phosphorylation via cGMP/PKG as a post-translational activation switch for CBS, coupling receptor signaling to H2S output and tissue relaxation.","evidence":"Site-specific phosphorylation analysis, CBS inhibition, and functional bladder strip and T24 cell assays","pmids":["27509878"],"confidence":"Medium","gaps":["Direct kinase-substrate biochemistry on Ser227 not reconstituted","Effect on enzyme kinetics not quantified"]},{"year":2019,"claim":"Established a mitochondrial CBS pool whose H2S tonically suppresses Complex IV, linking CBS overdosage to the mitochondrial and cognitive phenotypes of Down syndrome.","evidence":"Subcellular fractionation, CBS inhibition/siRNA with Complex IV and respiration assays in DS fibroblasts; Cbs KO/OE mice with behavioral and proteomic analysis","pmids":["31481613","30649339"],"confidence":"High","gaps":["Import mechanism targeting CBS to mitochondria undefined","Molecular targets of mitochondrial H2S beyond Complex IV not enumerated"]},{"year":2019,"claim":"Extended CBS regulation to NRF2-driven transcription and broadened its physiological reach to ferroptosis resistance and erythropoiesis/heme homeostasis.","evidence":"CBS/NRF2 RNAi and overexpression with ferroptosis/ROS readouts; CBS KO mouse heme-pathway and iron analysis","pmids":["31819185","31551410"],"confidence":"Medium","gaps":["Direct NRF2 binding at CBS promoter not shown in this entry","Mechanism linking CBS to ALAS2/FECH regulation unresolved"]},{"year":2020,"claim":"Revealed bidirectional coupling between CBS and the circadian clock through physical interaction with CRY1, integrating sulfur metabolism with circadian timekeeping.","evidence":"Co-IP, circadian period assays, CBS activity rescue in Cry1-/- liver, and transgenic mouse metabolomics","pmids":["32383312"],"confidence":"High","gaps":["Interaction interface and stoichiometry not mapped","Whether CRY1 binding alters CBS oligomerization unknown"]},{"year":2021,"claim":"Identified protein S-sulfhydration as the effector mechanism of CBS-derived H2S, with HuR and phosphodiesterases as direct targets controlling mRNA stability and cAMP/PKA-driven steroidogenesis.","evidence":"Biotin switch sulfhydration assays, CBS CRISPR KO and KO mouse models, mRNA chase, and PKA pathway analysis","pmids":["36725190","33713531"],"confidence":"High","gaps":["Sulfhydrated cysteine residues on targets not all mapped","Selectivity of CBS-derived vs other sulfide sources unclear"]},{"year":2021,"claim":"Linked CBS loss to genome-wide epigenetic remodeling resembling gastric cancer methylation patterns, expanding CBS function from metabolism to DNA methylation and inflammation control.","evidence":"CRISPR CBS deletion in gastric epithelial cells, global methylation profiling, NF-κB activity and H2S assays, proteomics","pmids":["34074348"],"confidence":"Medium","gaps":["Causal link from H2S to specific methylation changes not established","Whether NF-κB is cause or consequence unresolved"]},{"year":2023,"claim":"Demonstrated ATF6 as a direct transcriptional driver of CBS via promoter binding, channeling ER-stress signaling into H2S-mediated SIRT1 sulfhydration and metabolic control in liver.","evidence":"ATF6 liver KO mice, ChIP at CBS promoter, H2S and SIRT1 sulfhydration assays in high-fat diet model","pmids":["38007457"],"confidence":"Medium","gaps":["SIRT1 sulfhydration site not defined","Quantitative contribution to lipid phenotype unclear"]},{"year":2024,"claim":"Defined an ATF3-CBS axis sustaining ferroptosis resistance through TCA-cycle support under cystine deprivation, reinforcing CBS as a stress-adaptive transcriptional target in cancer.","evidence":"ATF3/CBS gain- and loss-of-function, ferroptosis and TCA metabolic assays, CRC patient correlation","pmids":["38490069"],"confidence":"Medium","gaps":["Direct ATF3 binding at CBS promoter not shown","Metabolic node connecting CBS to TCA flux not pinpointed"]},{"year":null,"claim":"How the diverse transcriptional inputs (NRF2, ATF3, ATF6, NF-κB), post-translational switches (Ser227 phosphorylation, CRY1 binding), and subcellular partitioning are integrated to set CBS-derived H2S output and target selection in a given cell remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model coupling transcriptional, allosteric, and PTM regulation","Determinants of mitochondrial vs cytosolic CBS targeting unknown","Rules governing which proteins are S-sulfhydrated not established"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016829","term_label":"lyase activity","supporting_discovery_ids":[3,22]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[16,17,19]},{"term_id":"GO:0140097","term_label":"catalytic activity, acting on DNA","supporting_discovery_ids":[18]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[8]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[22,23]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[23,6]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[3,12]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[10,20]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[10,20]},{"term_id":"R-HSA-9909396","term_label":"Circadian clock","supporting_discovery_ids":[8]}],"complexes":[],"partners":["CRY1","HUR","PDE4A","PDE8A","SIRT1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P35520","full_name":"Cystathionine beta-synthase","aliases":["Beta-thionase","Serine sulfhydrase"],"length_aa":551,"mass_kda":60.6,"function":"Hydro-lyase catalyzing the first step of the transsulfuration pathway, where the hydroxyl group of L-serine is displaced by L-homocysteine in a beta-replacement reaction to form L-cystathionine, the precursor of L-cysteine. This catabolic route allows the elimination of L-methionine and the toxic metabolite L-homocysteine (PubMed:20506325, PubMed:23974653, PubMed:23981774). 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disease","url":"https://pubmed.ncbi.nlm.nih.gov/31551410","citation_count":17,"is_preprint":false},{"pmid":"12413583","id":"PMC_12413583","title":"CBS 844ins68, MTHFR TT677 and EPCR 4031ins23 genotypes in patients with deep-vein thrombosis.","date":"2002","source":"Thrombosis research","url":"https://pubmed.ncbi.nlm.nih.gov/12413583","citation_count":17,"is_preprint":false},{"pmid":"28384716","id":"PMC_28384716","title":"Analysis of MTHFR, CBS, Glutathione, Taurine, and Hydrogen Sulfide Levels in Retinas of Hyperhomocysteinemic Mice.","date":"2017","source":"Investigative ophthalmology & visual science","url":"https://pubmed.ncbi.nlm.nih.gov/28384716","citation_count":17,"is_preprint":false},{"pmid":"23954866","id":"PMC_23954866","title":"Gene-environment and gene-gene interactions of specific MTHFR, MTR and CBS gene variants in relation to homocysteine in black South Africans.","date":"2013","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/23954866","citation_count":17,"is_preprint":false},{"pmid":"28881655","id":"PMC_28881655","title":"DNA hypomethylation of CBS promoter induced by folate deficiency is a potential noninvasive circulating biomarker for colorectal adenocarcinomas.","date":"2017","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/28881655","citation_count":17,"is_preprint":false},{"pmid":"36701996","id":"PMC_36701996","title":"Total flavones of Rhododendron induce the transformation of A1/A2 astrocytes via promoting the release of CBS-produced H2S.","date":"2023","source":"Phytomedicine : international journal of phytotherapy and phytopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/36701996","citation_count":17,"is_preprint":false},{"pmid":"34795411","id":"PMC_34795411","title":"ADT-OH inhibits malignant melanoma metastasis in mice via suppressing CSE/CBS and FAK/Paxillin signaling pathway.","date":"2021","source":"Acta pharmacologica Sinica","url":"https://pubmed.ncbi.nlm.nih.gov/34795411","citation_count":16,"is_preprint":false},{"pmid":"35588172","id":"PMC_35588172","title":"Rutin Inhibits the Progression of Osteoarthritis Through CBS-Mediated RhoA/ROCK Signaling.","date":"2022","source":"DNA and cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/35588172","citation_count":16,"is_preprint":false},{"pmid":"21520339","id":"PMC_21520339","title":"Identification and functional analyses of CBS alleles in Spanish and Argentinian homocystinuric patients.","date":"2011","source":"Human mutation","url":"https://pubmed.ncbi.nlm.nih.gov/21520339","citation_count":16,"is_preprint":false},{"pmid":"32778975","id":"PMC_32778975","title":"Genome-Wide Investigation and Expression Profiling Under Abiotic Stresses of a Soybean Unknown Function (DUF21) and Cystathionine-β-Synthase (CBS) Domain-Containing Protein Family.","date":"2020","source":"Biochemical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/32778975","citation_count":16,"is_preprint":false},{"pmid":"30023284","id":"PMC_30023284","title":"S-adenosylhomocysteine hydrolase over-expression does not alter S-adenosylmethionine or S-adenosylhomocysteine levels in CBS deficient mice.","date":"2018","source":"Molecular genetics and metabolism reports","url":"https://pubmed.ncbi.nlm.nih.gov/30023284","citation_count":16,"is_preprint":false},{"pmid":"18242101","id":"PMC_18242101","title":"Molecular gene cloning and overexpression of the phytase from Debaryomyces castellii CBS 2923.","date":"2008","source":"Protein expression and purification","url":"https://pubmed.ncbi.nlm.nih.gov/18242101","citation_count":16,"is_preprint":false},{"pmid":"17336565","id":"PMC_17336565","title":"DNA methylation status is not impaired in treated cystathionine beta-synthase (CBS) deficient patients.","date":"2007","source":"Molecular genetics and metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/17336565","citation_count":16,"is_preprint":false},{"pmid":"26400082","id":"PMC_26400082","title":"Cystathionine β-Synthase (CBS) Domain-containing Pyrophosphatase as a Target for Diadenosine Polyphosphates in Bacteria.","date":"2015","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/26400082","citation_count":16,"is_preprint":false},{"pmid":"35315960","id":"PMC_35315960","title":"Homozygous whole body Cbs knockout in adult mice features minimal pathology during ageing despite severe homocysteinemia.","date":"2022","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/35315960","citation_count":15,"is_preprint":false},{"pmid":"36646297","id":"PMC_36646297","title":"Sodium hydrosulfide inhibits hemin-induced ferroptosis and lipid peroxidation in BV2 cells via the CBS/H2S system.","date":"2023","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/36646297","citation_count":15,"is_preprint":false},{"pmid":"33713531","id":"PMC_33713531","title":"H2 S catalysed by CBS regulates testosterone synthesis through affecting the sulfhydrylation of PDE.","date":"2021","source":"Journal of cellular and molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/33713531","citation_count":15,"is_preprint":false},{"pmid":"30030379","id":"PMC_30030379","title":"Mouse modeling and structural analysis of the p.G307S mutation in human cystathionine β-synthase (CBS) reveal effects on CBS activity but not stability.","date":"2018","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/30030379","citation_count":15,"is_preprint":false},{"pmid":"32800907","id":"PMC_32800907","title":"Association of maternal dietary intakes and CBS gene polymorphisms with congenital heart disease in offspring.","date":"2020","source":"International journal of cardiology","url":"https://pubmed.ncbi.nlm.nih.gov/32800907","citation_count":14,"is_preprint":false},{"pmid":"33486070","id":"PMC_33486070","title":"Acute acrylonitrile exposure inhibits endogenous H2S biosynthesis in rat brain and liver: The role of CBS/3-MPST-H2S pathway in its astrocytic toxicity.","date":"2021","source":"Toxicology","url":"https://pubmed.ncbi.nlm.nih.gov/33486070","citation_count":14,"is_preprint":false},{"pmid":"29508359","id":"PMC_29508359","title":"Eight novel mutations of CBS gene in nine Chinese patients with classical homocystinuria.","date":"2018","source":"World journal of pediatrics : WJP","url":"https://pubmed.ncbi.nlm.nih.gov/29508359","citation_count":14,"is_preprint":false},{"pmid":"22512572","id":"PMC_22512572","title":"Role of polymorphisms in factor V (FV Leiden), prothrombin, plasminogen activator inhibitor type-1 (PAI-1), methylenetetrahydrofolate reductase (MTHFR) and cystathionine β-synthase (CBS) genes as risk factors for thrombophilias.","date":"2012","source":"Mini reviews in medicinal chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/22512572","citation_count":14,"is_preprint":false},{"pmid":"20934423","id":"PMC_20934423","title":"The CBS domain protein MJ0729 of Methanocaldococcus jannaschii binds DNA.","date":"2010","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/20934423","citation_count":13,"is_preprint":false},{"pmid":"27444757","id":"PMC_27444757","title":"Lack of global epigenetic methylation defects in CBS deficient mice.","date":"2016","source":"Journal of inherited metabolic disease","url":"https://pubmed.ncbi.nlm.nih.gov/27444757","citation_count":13,"is_preprint":false},{"pmid":"9525476","id":"PMC_9525476","title":"Expression of TGFalpha autocrine activity in human colon carcinoma CBS cells is autoregulated and independent of exogenous epidermal growth factor.","date":"1998","source":"Journal of cellular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/9525476","citation_count":13,"is_preprint":false},{"pmid":"38007457","id":"PMC_38007457","title":"Activating transcription factor (ATF) 6 upregulates cystathionine β synthetase (CBS) expression and hydrogen sulfide (H2S) synthesis to ameliorate liver metabolic damage.","date":"2023","source":"European journal of medical research","url":"https://pubmed.ncbi.nlm.nih.gov/38007457","citation_count":12,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":53164,"output_tokens":5979,"usd":0.124589,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":15131,"output_tokens":5348,"usd":0.104678,"stage2_stop_reason":"end_turn"},"total_usd":0.229267,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2004,\n      \"finding\": \"Tandem pairs of CBS domains from cystathionine beta-synthase bind AMP, ATP, or S-adenosyl methionine; disease-causing mutations in the CBS domains impair this nucleotide binding, establishing CBS domains as adenosine-derivative sensing modules.\",\n      \"method\": \"In vitro binding assays with purified CBS domain proteins; functional assessment of disease mutants\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct biochemical binding assay with multiple proteins and disease mutant validation, replicated across multiple CBS-domain-containing proteins\",\n      \"pmids\": [\"14722619\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Human CBS contains two classes of SAM-binding sites in the C-terminal regulatory domain: a high-affinity set of two sites involved in kinetic stabilization, and a low-affinity set of four sites responsible for enzyme activation. SAM-induced stabilization modulates steady-state CBS levels in vivo.\",\n      \"method\": \"Calorimetric methods (ITC), functional enzyme assays, kinetic modeling on purified WT and homocystinuria mutant CBS proteins\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — multiple orthogonal methods (calorimetry + functional assays + kinetic modeling) in single lab on purified protein\",\n      \"pmids\": [\"22985361\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Eight homocystinuria-causing CBS missense mutants (P49L, P78R, A114V, R125Q, E176K, P422L, I435T, S466L) can be rescued by chemical chaperones during expression, yielding tetrameric enzymes with restored catalytic activity; R125Q and E176K fail to respond to SAM stimulation or thermal activation, indicating their conformations cannot reach the activated state.\",\n      \"method\": \"Purification of CBS mutant proteins expressed in E. coli with chemical chaperones; enzyme activity assays; thermal stability measurements; SAM stimulation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstitution with purified proteins, multiple orthogonal functional readouts, single lab\",\n      \"pmids\": [\"20308073\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"CBS catalyzes PLP-dependent reactions to produce cystathionine and H2S; the C. elegans CBS-1 ortholog has a unique tandem-repeat architecture with one active PLP-binding catalytic module and one inactive N-terminal module, lacks heme and the regulatory Bateman domain, forms monomers, and its knockdown causes ~10-fold elevation of homocysteine.\",\n      \"method\": \"Truncation and active-site mutagenesis of purified CBS-1; PLP-binding assays; in vivo RNAi knockdown; homocysteine measurement\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstitution with truncated/mutant proteins, multiple orthogonal methods (activity assay, mutagenesis, RNAi phenotype), single lab\",\n      \"pmids\": [\"22240119\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The p.G307S CBS mutation abolishes enzyme activity without affecting protein stability or multimerization; molecular dynamics simulation indicates the substitution restricts Y308 conformational flexibility required for formation of the pyridoxal-cystathionine catalytic intermediate.\",\n      \"method\": \"Transgenic mouse model expressing p.G307S CBS; enzyme activity assays; proteasome inhibitor treatment; molecular dynamics simulation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vivo mouse model plus in vitro biochemical characterization plus structural simulation, multiple methods in single lab\",\n      \"pmids\": [\"30030379\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Reduced SAM activation of CBS is caused by C-terminal domain mutations that produce abnormal oligomeric conformations unable to reach the activated state, while catalytic-core mutations reduce protein expression and activity; SAM-activation defect confirmed in both patient fibroblasts and recombinant mutant proteins.\",\n      \"method\": \"Retrospective fibroblast activity assays; heterologous CBS mutant expression in E. coli; Western blotting; enzyme activity in absence/presence of SAM and PLP\",\n      \"journal\": \"Journal of inherited metabolic disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — multiple mutant proteins characterized, but single lab, no structural validation\",\n      \"pmids\": [\"23974653\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CBS-derived H2S directly suppresses mitochondrial Complex IV activity; pharmacological inhibition of CBS (aminooxyacetate) or siRNA silencing in Down syndrome fibroblasts normalizes H2S levels, restores Complex IV activity, improves mitochondrial electron transport and ATP synthesis, and restores cell proliferation.\",\n      \"method\": \"CBS inhibition (aminooxyacetate) and siRNA knockdown in DS fibroblasts; measurement of H2S, polysulfides, mitochondrial oxygen consumption, ATP generation, Complex IV activity\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two orthogonal loss-of-function approaches (pharmacological + siRNA) with defined cellular and biochemical phenotype, single lab\",\n      \"pmids\": [\"31481613\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Cbs overdosage is necessary and sufficient for recognition-memory deficits in Down syndrome mouse models; Cbs interacts genetically (epistasis) with Dyrk1a to control memory, and proteomic analysis implicates synaptic transmission, cell projection morphogenesis, and actin cytoskeleton pathways.\",\n      \"method\": \"Genetic deletion and transgenic overexpression of Cbs in mice; novel object recognition behavioral testing; pharmacological rescue; genetic epistasis with Dyrk1a; proteomic analysis\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — necessity and sufficiency established by KO and OE, genetic epistasis, pharmacological rescue, and proteomics in single lab\",\n      \"pmids\": [\"30649339\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CBS physically interacts with the core circadian protein CRY1; in cells, CBS augments CRY1-mediated repression of the CLOCK/BMAL1 complex and shortens circadian period; CRY1 binding reciprocally modulates CBS enzymatic activity; liver extracts from Cry1-/- mice show reduced CBS activity restored by exogenous WT CRY1.\",\n      \"method\": \"Co-immunoprecipitation; circadian period assays; CBS enzymatic activity in Cry1-/- liver extracts with/without exogenous CRY1; metabolomics; transgenic CbsZn/Zn mouse circadian analysis\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal functional interaction demonstrated by Co-IP, enzyme activity rescue, and in vivo mouse model with metabolomics, single lab\",\n      \"pmids\": [\"32383312\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Muscarinic receptor (M1/M3) activation in human urothelium phosphorylates CBS at Ser227 via the cGMP/PKG pathway, stimulating H2S production and cGMP, which promotes bladder relaxation; CBS (but not CSE) inhibition mimics urothelium removal and increases carbachol-induced contraction.\",\n      \"method\": \"CBS inhibition pharmacology; site-specific phosphorylation analysis (Ser227); functional bladder strip assays; T24 cell line experiments; receptor blockade\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — post-translational modification (phosphorylation) identified with pathway context and functional consequence, single lab\",\n      \"pmids\": [\"27509878\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CBS-derived H2S production is activated by NRF2 transcriptional upregulation of CBS in erastin-resistant ovarian cancer cells; CBS knockdown causes ferroptotic cell death in resistant cells, while CBS overexpression confers resistance, demonstrating NRF2/CBS signaling as a ferroptosis resistance mechanism through the transsulfuration pathway.\",\n      \"method\": \"RNAi knockdown of CBS and NRF2; CBS overexpression; cell viability and ROS measurement; metabolite analysis of transsulfuration pathway\",\n      \"journal\": \"British journal of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — bidirectional genetic manipulation (KD and OE) with defined ferroptosis phenotype, single lab\",\n      \"pmids\": [\"31819185\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CBS regulates mitochondrial bioenergetics in ovarian cancer cells by controlling ROS production, oxygen consumption, and ATP generation; CBS silencing via nanoliposomal siRNA inhibits tumor growth and sensitizes cisplatin-resistant cells.\",\n      \"method\": \"CBS siRNA silencing (in vitro and in vivo); mitochondrial ROS, oxygen consumption, ATP measurements; orthotopic mouse model\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function in vitro and in vivo with multiple bioenergetic readouts, single lab\",\n      \"pmids\": [\"24236104\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"SAM, as an allosteric CBS activator, markedly enhances CBS-mediated H2S production in vitro; low-to-intermediate SAM concentrations stimulate HCT116 colon cancer cell proliferation and bioenergetics in a CBS-dependent manner (attenuated by AOAA or CBS silencing), while high SAM concentrations inhibit proliferation by CBS-independent mechanisms.\",\n      \"method\": \"Recombinant CBS enzyme assay; HCT116 cell proliferation (xCELLigence); extracellular flux bioenergetics; CBS stable silencing; AOAA pharmacological inhibition\",\n      \"journal\": \"Nitric oxide : biology and chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzyme assay plus cell-based pharmacological and genetic validation, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"24667534\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TLR4 activation upregulates CBS expression in dorsal root ganglion neurons via NF-κB signaling; NF-κB inhibition reduces CBS upregulation and attenuates visceral hypersensitivity in a rat IBS model.\",\n      \"method\": \"Western blot; intrathecal pharmacological inhibition of TLR4 and NF-κB; LPS stimulation of cultured DRG neurons; patch-clamp electrophysiology\",\n      \"journal\": \"World journal of gastroenterology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological and in vitro manipulation defining upstream pathway to CBS, single lab, multiple readouts\",\n      \"pmids\": [\"26229403\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Serotonin and dopamine increase CBS expression via mTOR signaling and stimulate H2S production in rat smooth muscle cells, protecting against hypothermia/rewarming-induced ROS and apoptosis; CBS inhibition (pharmacological or siRNA) abolishes these protective effects.\",\n      \"method\": \"CBS mRNA/protein measurement; siRNA knockdown; pharmacological CBS inhibition; ROS measurement; caspase activity assay; organ preservation experiments\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two orthogonal CBS loss-of-function methods with defined cytoprotective phenotype, single lab\",\n      \"pmids\": [\"21829469\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Estradiol-17β (E2) specifically stimulates CBS (but not CSE) expression and H2S production in endometrial stromal cells via estrogen receptor (blocked by ICI 182780); stromal CBS-derived H2S promotes endometrial microvascular endothelial cell angiogenesis in a paracrine manner.\",\n      \"method\": \"CBS/CSE expression analysis; ICI 182780 receptor blockade; CBS pharmacological inhibition; co-culture migration assay; in vivo endometrial analysis across hormonal states\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — receptor-specific regulation and functional consequence demonstrated with pharmacological inhibition, single lab\",\n      \"pmids\": [\"32987401\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"H2S produced by CBS promotes S-sulfhydration of HuR (human antigen R), which decreases HuR binding to COX-2 mRNA, reducing COX-2 mRNA stability and expression; CBS heterozygous knockout mice show decreased HuR sulfhydration and increased COX-2 in colon.\",\n      \"method\": \"Biotin switch technique for S-sulfhydration; CRISPR/Cas9 CBS knockout; ActD chase assay for mRNA stability; CBS+/- mouse colitis model; immunofluorescence\",\n      \"journal\": \"Journal of advanced research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct PTM (sulfhydration) identified with functional consequence on mRNA stability, validated in vivo, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"36725190\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CBS-derived H2S promotes testosterone synthesis in MLTC-1 cells by sulfhydrylating PDE4A and PDE8A (reducing their activity), thereby elevating cAMP and activating PKA pathway; CBS overexpression increases StAR, P450scc, P450c17 and 3β-HSD expression.\",\n      \"method\": \"CBS overexpression/knockdown; CBS/H2S measurement; biotin switch technique for PDE sulfhydrylation; PKA pathway analysis; RT-qPCR and western blot\",\n      \"journal\": \"Journal of cellular and molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct PTM (sulfhydration) of substrate PDE identified by biotin switch, functional pathway consequence demonstrated, single lab\",\n      \"pmids\": [\"33713531\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CBS knockout using CRISPR in normal gastric epithelial cells induces widespread DNA methylation changes overlapping with gastric cancer CIMP patterns, and CBS loss reduces H2S and increases NF-κB activity, linking CBS to both epigenetic regulation and inflammation.\",\n      \"method\": \"CRISPR deletion of CBS; global DNA methylation profiling; NF-κB activity assay; H2S measurement; proteomics; comparison with primary gastric cancer CIMP patterns\",\n      \"journal\": \"Genome biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR KO with defined molecular phenotypes (methylation, NF-κB), single lab\",\n      \"pmids\": [\"34074348\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ATF6 directly binds the CBS gene promoter and transcriptionally upregulates CBS expression; ATF6-driven CBS activity increases H2S synthesis, which sulfhydrates SIRT1, reducing lipogenesis and inflammation in fatty liver.\",\n      \"method\": \"ATF6 liver-specific knockout mice; chromatin immunoprecipitation (ChIP) at CBS promoter; H2S measurement; SIRT1 sulfhydration assay; high-fat diet model\",\n      \"journal\": \"European journal of medical research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP establishes direct ATF6 binding at CBS promoter with downstream H2S/SIRT1 consequence, single lab\",\n      \"pmids\": [\"38007457\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ATF3 positively regulates CBS expression under cystine deprivation, conferring ferroptosis resistance in colorectal cancer; CBS suppression sensitizes cells to ferroptosis by targeting the mitochondrial TCA cycle; ATF3-CBS axis positively correlates with CRC progression.\",\n      \"method\": \"ATF3 and CBS gain/loss-of-function; ferroptosis assays; metabolic (TCA cycle) analysis; CRC patient correlation\",\n      \"journal\": \"Redox biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — bidirectional genetic manipulation of ATF3-CBS axis with defined ferroptosis and metabolic readouts, single lab\",\n      \"pmids\": [\"38490069\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CBS deficiency suppresses erythropoiesis by reducing expression of heme biosynthetic enzymes ALAS2 and FECH, heme exporter FLVCR, HIF-2α, EPO, and EPOR in CBS knockout mice, while increasing IL-6, hepcidin, and iron content.\",\n      \"method\": \"CBS-/-, CBS+/-, and CBS+/+ mouse comparison; Western blot and qPCR for heme pathway proteins; blood and bone marrow iron content measurement\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with defined molecular phenotype across multiple pathway components, single lab\",\n      \"pmids\": [\"31551410\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"CBS protein is localized predominantly in the cytosol of neurons, with strongest expression in Purkinje cell bodies/neuronal processes and Ammon's horn neurons in adult mouse brain; CBS expression peaks during cerebellar development and is present in liver from early embryonic stages.\",\n      \"method\": \"In situ hybridization; Northern blotting; immunohistochemistry in mouse brain and developmental tissues\",\n      \"journal\": \"The journal of histochemistry and cytochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct subcellular/tissue localization by immunohistochemistry and in situ hybridization, single lab, no functional consequence linked\",\n      \"pmids\": [\"12588964\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CBS localizes to both cytosol and mitochondria in Down syndrome fibroblasts, and mitochondrial CBS-derived H2S is responsible for tonic suppression of Complex IV.\",\n      \"method\": \"CBS subcellular fractionation and localization in DS fibroblasts; CBS inhibition/siRNA with Complex IV activity measurement\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct fractionation establishing mitochondrial localization with functional consequence on Complex IV, single lab\",\n      \"pmids\": [\"31481613\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"A 31 bp VNTR spanning the exon 13-intron 13 boundary of the CBS gene is associated with reduced CBS enzyme activity in patient fibroblasts (measured directly) and elevated post-methionine load homocysteine; RT-PCR shows evidence of alternative splicing at this junction dependent on repeat number.\",\n      \"method\": \"CBS enzyme activity in cultured fibroblasts; RT-PCR; VNTR genotyping in patients and controls\",\n      \"journal\": \"European journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct enzyme activity measurement in primary fibroblasts correlating with genotype, with RT-PCR evidence of splicing mechanism, single lab\",\n      \"pmids\": [\"11528503\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"CBS (alongside CSE) is expressed in adrenocortical cells and is required for H2S generation; CBS/CSE inhibition or siRNA knockdown causes mitochondrial oxidative stress and dysfunction and blunts corticosterone responses to ACTH; LPS inhibits CBS/CSE expression and H2S production in adrenal glands.\",\n      \"method\": \"CBS/CSE inhibitors and siRNA in adrenocortical cells; in vivo adrenal suppression model; mitochondrial function assays; corticosterone measurement\",\n      \"journal\": \"Antioxidants & redox signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two orthogonal loss-of-function approaches in vitro and in vivo with defined steroidogenic phenotype, single lab\",\n      \"pmids\": [\"24702258\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CBS (cystathionine β-synthase) is a PLP- and heme-containing tetrameric enzyme that catalyzes the first committed step of the transsulfuration pathway (condensation of serine and homocysteine to cystathionine) and produces H2S; its activity is allosterically activated by SAM binding to two classes of sites in the C-terminal regulatory (Bateman/CBS) domain, it is post-translationally regulated by cGMP/PKG-dependent phosphorylation at Ser227 and by interaction with the circadian protein CRY1, it localizes to both cytosol and mitochondria where CBS-derived H2S tonically suppresses Complex IV, it acts as a transcriptional target of NRF2, ATF3, ATF6, and NF-κB, and it promotes ferroptosis resistance and cancer cell bioenergetics through H2S-mediated sulfhydration of downstream targets including HuR and phosphodiesterases.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CBS (cystathionine β-synthase) is a PLP-dependent enzyme that catalyzes the condensation of serine and homocysteine to cystathionine and concomitantly generates hydrogen sulfide (H2S), positioning it as the entry point of the transsulfuration pathway and a major source of signaling sulfide [#3]. Enzyme output is set by allosteric control through the C-terminal regulatory (Bateman/CBS) domain, which binds adenosine derivatives including AMP, ATP, and S-adenosylmethionine; SAM occupies two classes of sites — high-affinity sites that kinetically stabilize the protein and modulate its steady-state level, and low-affinity sites that drive catalytic activation [#0, #1, #12]. Homocystinuria-causing missense mutations act by distinct routes: catalytic-core substitutions reduce activity or expression (and one, G307S, restricts the conformational flexibility needed to form the catalytic intermediate), whereas C-terminal mutations trap abnormal oligomers that cannot reach the SAM-activated state, defects partially correctable by chemical chaperones [#2, #4, #5]. Beyond intrinsic regulation, CBS is post-translationally controlled by cGMP/PKG-dependent phosphorylation at Ser227 and by physical interaction with the circadian repressor CRY1, which reciprocally modulates CBS activity while CBS augments CRY1-mediated repression of CLOCK/BMAL1 [#8, #9]. CBS is a transcriptional target of multiple stress and inflammatory programs — NRF2, ATF3, ATF6, and NF-κB — that converge to raise CBS-derived H2S [#10, #13, #19, #20]. Downstream, this H2S acts through protein S-sulfhydration of targets including HuR (destabilizing COX-2 mRNA), phosphodiesterases PDE4A/PDE8A (elevating cAMP/PKA signaling for steroidogenesis), and SIRT1 (limiting hepatic lipogenesis), and it tonically suppresses mitochondrial Complex IV from a mitochondrial pool of the enzyme [#16, #17, #19, #23]. Functionally, CBS-driven sulfide supports cancer cell bioenergetics and ferroptosis resistance, modulates erythropoiesis and the heme pathway, and contributes to cytoprotection and angiogenesis across tissues [#10, #11, #20, #21].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Established a genetic determinant of CBS activity by linking a regulatory-region VNTR to reduced enzyme function, connecting sequence variation to the transsulfuration phenotype.\",\n      \"evidence\": \"CBS enzyme activity in patient fibroblasts, RT-PCR splicing analysis, and VNTR genotyping\",\n      \"pmids\": [\"11528503\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking repeat number to splicing outcome not fully resolved\", \"Effect size on clinical phenotype unclear\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Defined the tissue and subcellular distribution of CBS, showing predominantly cytosolic neuronal expression with developmental regulation, framing where the enzyme acts.\",\n      \"evidence\": \"In situ hybridization, Northern blot, and immunohistochemistry in mouse brain and embryonic tissues\",\n      \"pmids\": [\"12588964\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No functional consequence linked to localization\", \"Mitochondrial pool not yet identified\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identified the CBS domains as adenosine-derivative sensing modules, explaining how nucleotide ligands and disease mutations converge on the enzyme's regulatory region.\",\n      \"evidence\": \"In vitro binding assays with purified CBS-domain proteins and disease-mutant functional tests\",\n      \"pmids\": [\"14722619\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative coupling of ligand binding to catalytic rate not defined\", \"Structural basis of mutant binding loss not resolved here\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Resolved the dual logic of SAM regulation — distinguishing high-affinity stabilizing sites that set protein level from low-affinity activating sites — explaining how a single metabolite both stabilizes and activates CBS.\",\n      \"evidence\": \"ITC, functional enzyme assays, and kinetic modeling on purified WT and homocystinuria-mutant CBS\",\n      \"pmids\": [\"22985361\", \"23974653\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural geometry of the six sites not directly visualized\", \"In vivo contribution of stabilization vs activation hard to disentangle\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Dissected the conserved catalytic core through a divergent ortholog lacking heme and the Bateman domain, isolating the PLP-dependent module responsible for cystathionine/H2S production and homocysteine homeostasis.\",\n      \"evidence\": \"Truncation/active-site mutagenesis of C. elegans CBS-1, PLP-binding assays, and RNAi knockdown with homocysteine measurement\",\n      \"pmids\": [\"22240119\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ortholog lacks the regulatory features of human CBS\", \"Heme contribution to catalysis not addressed\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Classified homocystinuria mutations mechanistically and showed chemical chaperones can rescue folding-defective mutants, separating conformational defects from irreversibly inactivated states.\",\n      \"evidence\": \"Purified CBS mutants expressed with chemical chaperones, activity, thermal stability, and SAM-stimulation assays\",\n      \"pmids\": [\"20308073\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo chaperone efficacy not tested here\", \"R125Q/E176K activation failure structurally unexplained\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Pinpointed a single-residue catalytic mechanism by which G307S abolishes activity without destabilizing the protein, isolating conformational flexibility at the active site as essential for intermediate formation.\",\n      \"evidence\": \"Transgenic G307S mouse, enzyme assays, proteasome inhibition, and molecular dynamics simulation\",\n      \"pmids\": [\"30030379\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Simulation prediction not confirmed by crystallography\", \"Generalizability to other core mutations untested\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Linked extracellular signals to CBS-dependent cytoprotection, showing monoamine/mTOR-driven CBS expression generates H2S that limits oxidative apoptosis.\",\n      \"evidence\": \"CBS mRNA/protein measurement, siRNA and pharmacological inhibition, ROS and caspase assays in smooth muscle cells\",\n      \"pmids\": [\"21829469\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct mTOR-to-CBS transcriptional link not mapped\", \"H2S effector targets not identified\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Connected CBS-derived H2S to cancer bioenergetics, demonstrating that SAM-activated CBS fuels tumor proliferation, mitochondrial respiration, and chemoresistance.\",\n      \"evidence\": \"Recombinant enzyme assays, CBS silencing in vitro/in vivo, bioenergetic flux measurements, and orthotopic tumor model\",\n      \"pmids\": [\"24667534\", \"24236104\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Biphasic SAM effect mechanism (CBS-independent at high dose) unresolved\", \"Direct sulfhydration targets in this context not identified\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identified Ser227 phosphorylation via cGMP/PKG as a post-translational activation switch for CBS, coupling receptor signaling to H2S output and tissue relaxation.\",\n      \"evidence\": \"Site-specific phosphorylation analysis, CBS inhibition, and functional bladder strip and T24 cell assays\",\n      \"pmids\": [\"27509878\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct kinase-substrate biochemistry on Ser227 not reconstituted\", \"Effect on enzyme kinetics not quantified\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Established a mitochondrial CBS pool whose H2S tonically suppresses Complex IV, linking CBS overdosage to the mitochondrial and cognitive phenotypes of Down syndrome.\",\n      \"evidence\": \"Subcellular fractionation, CBS inhibition/siRNA with Complex IV and respiration assays in DS fibroblasts; Cbs KO/OE mice with behavioral and proteomic analysis\",\n      \"pmids\": [\"31481613\", \"30649339\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Import mechanism targeting CBS to mitochondria undefined\", \"Molecular targets of mitochondrial H2S beyond Complex IV not enumerated\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Extended CBS regulation to NRF2-driven transcription and broadened its physiological reach to ferroptosis resistance and erythropoiesis/heme homeostasis.\",\n      \"evidence\": \"CBS/NRF2 RNAi and overexpression with ferroptosis/ROS readouts; CBS KO mouse heme-pathway and iron analysis\",\n      \"pmids\": [\"31819185\", \"31551410\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct NRF2 binding at CBS promoter not shown in this entry\", \"Mechanism linking CBS to ALAS2/FECH regulation unresolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Revealed bidirectional coupling between CBS and the circadian clock through physical interaction with CRY1, integrating sulfur metabolism with circadian timekeeping.\",\n      \"evidence\": \"Co-IP, circadian period assays, CBS activity rescue in Cry1-/- liver, and transgenic mouse metabolomics\",\n      \"pmids\": [\"32383312\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Interaction interface and stoichiometry not mapped\", \"Whether CRY1 binding alters CBS oligomerization unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identified protein S-sulfhydration as the effector mechanism of CBS-derived H2S, with HuR and phosphodiesterases as direct targets controlling mRNA stability and cAMP/PKA-driven steroidogenesis.\",\n      \"evidence\": \"Biotin switch sulfhydration assays, CBS CRISPR KO and KO mouse models, mRNA chase, and PKA pathway analysis\",\n      \"pmids\": [\"36725190\", \"33713531\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Sulfhydrated cysteine residues on targets not all mapped\", \"Selectivity of CBS-derived vs other sulfide sources unclear\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Linked CBS loss to genome-wide epigenetic remodeling resembling gastric cancer methylation patterns, expanding CBS function from metabolism to DNA methylation and inflammation control.\",\n      \"evidence\": \"CRISPR CBS deletion in gastric epithelial cells, global methylation profiling, NF-κB activity and H2S assays, proteomics\",\n      \"pmids\": [\"34074348\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal link from H2S to specific methylation changes not established\", \"Whether NF-κB is cause or consequence unresolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Demonstrated ATF6 as a direct transcriptional driver of CBS via promoter binding, channeling ER-stress signaling into H2S-mediated SIRT1 sulfhydration and metabolic control in liver.\",\n      \"evidence\": \"ATF6 liver KO mice, ChIP at CBS promoter, H2S and SIRT1 sulfhydration assays in high-fat diet model\",\n      \"pmids\": [\"38007457\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"SIRT1 sulfhydration site not defined\", \"Quantitative contribution to lipid phenotype unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined an ATF3-CBS axis sustaining ferroptosis resistance through TCA-cycle support under cystine deprivation, reinforcing CBS as a stress-adaptive transcriptional target in cancer.\",\n      \"evidence\": \"ATF3/CBS gain- and loss-of-function, ferroptosis and TCA metabolic assays, CRC patient correlation\",\n      \"pmids\": [\"38490069\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ATF3 binding at CBS promoter not shown\", \"Metabolic node connecting CBS to TCA flux not pinpointed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the diverse transcriptional inputs (NRF2, ATF3, ATF6, NF-κB), post-translational switches (Ser227 phosphorylation, CRY1 binding), and subcellular partitioning are integrated to set CBS-derived H2S output and target selection in a given cell remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model coupling transcriptional, allosteric, and PTM regulation\", \"Determinants of mitochondrial vs cytosolic CBS targeting unknown\", \"Rules governing which proteins are S-sulfhydrated not established\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016829\", \"supporting_discovery_ids\": [3, 22]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [16, 17, 19]},\n      {\"term_id\": \"GO:0140097\", \"supporting_discovery_ids\": [18]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [22, 23]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [23, 6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [3, 12]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [10, 20]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [10, 20]},\n      {\"term_id\": \"R-HSA-9909396\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CRY1\", \"HuR\", \"PDE4A\", \"PDE8A\", \"SIRT1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}