{"gene":"GUCY1A1","run_date":"2026-04-28T18:06:53","timeline":{"discoveries":[{"year":2016,"finding":"GUCY1A3 encodes the α1 subunit of soluble guanylate cyclase (sGC); a missense variant (Cys517Tyr) in GUCY1A3 produces a mutant α1 protein with significantly blunted cGMP signaling response upon nitric oxide (NO) exposure, establishing loss-of-function as the mechanism linking GUCY1A3 mutations to moyamoya disease and hypertension.","method":"Biochemical functional assay of mutant protein signaling response to NO donor in vitro","journal":"Clinical genetics","confidence":"Medium","confidence_rationale":"Tier 2 — direct functional assay of mutant protein, single lab/study","pmids":["26777256"],"is_preprint":false},{"year":2014,"finding":"A missense variant α1-A680T in GUCY1A3 results in higher cGMP production in reporter cells and enhanced sensitivity to nitric oxide in purified protein in vitro, conferring a gain-of-function phenotype protective against high-altitude pulmonary hypertension.","method":"Reporter cell cGMP assay and in vitro enzymatic assay of purified α1-A680T sGC","journal":"Circulation. Cardiovascular genetics","confidence":"Medium","confidence_rationale":"Tier 1 — in vitro enzymatic assay with purified protein and cell-based assay, single study","pmids":["25373139"],"is_preprint":false},{"year":2016,"finding":"Rare coding GUCY1A3 variants found in MI patients all dimerize with the β1 subunit (co-immunoprecipitation); five variants display decreased cGMP production upon NO stimulation, and this reduced activity is rescued by the sGC stimulator BAY 41-2272 in vitro.","method":"Co-immunoprecipitation for dimerization; cGMP radioimmunoassay after NO donor stimulation in HEK293 cells; pharmacological rescue with BAY 41-2272","journal":"Basic research in cardiology","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods (Co-IP, enzymatic assay, pharmacological rescue) in single study","pmids":["27342234"],"is_preprint":false},{"year":2017,"finding":"The transcription factor ZEB1 binds preferentially to the non-risk allele of rs7692387 (intronic site), driving higher GUCY1A3 promoter activity and expression; ZEB1 knockdown reduces non-risk allele promoter activity and endogenous GUCY1A3 expression. Higher sGC (α1) levels lead to enhanced cGMP-mediated inhibition of ADP-induced platelet aggregation and reduced vascular smooth muscle cell migration upon pharmacological sGC stimulation.","method":"Allele-specific ChIP; siRNA knockdown and overexpression of ZEB1; reporter gene assays; platelet aggregation assay ex vivo; VSMC migration assay","journal":"Circulation","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (ChIP, siRNA, reporter assay, functional cell assays), replicated in human ex vivo samples","pmids":["28487391"],"is_preprint":false},{"year":2004,"finding":"Antisense knockdown of GUCY1A3 (or GUCY1B3) in glioma cell lines markedly reduced cGMP content and VEGF expression, inhibited HUVEC growth in vitro, and suppressed subcutaneous tumor vascularization in vivo, establishing that GUCY1A3-driven cGMP production is an upstream mediator of VEGF expression and angiogenesis in glioma.","method":"Antisense RNA transfection; cGMP measurement; VEGF expression assay; HUVEC co-culture angiogenesis assay; nude mouse tumor model with vascular index quantification","journal":"Oncology reports","confidence":"Medium","confidence_rationale":"Tier 2 — loss-of-function with multiple phenotypic readouts in vitro and in vivo, single lab","pmids":["15201957"],"is_preprint":false},{"year":2023,"finding":"Endothelial cell-specific knockout of GUCY1A1 in mice increases microvascular no-reflow area, infarction size, and cardiac dysfunction after ischemia-reperfusion injury; mechanistically, PKG (downstream of sGC-cGMP) phosphorylates LDHA at threonine 95, activating LDHA's moonlighting kinase function to phosphorylate GPX4 at serine 131, reducing chaperone-mediated autophagy-dependent GPX4 degradation and thereby suppressing ferroptosis in endothelial cells.","method":"EC-specific conditional knockout and AAV-mediated overexpression in mice; mass spectrometry identification of phosphorylation sites; CRISPR-Cas9 mutagenesis of phosphorylation sites; co-immunoprecipitation for protein interactions; chaperone-mediated autophagy assay","journal":"Circulation research","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods including KO/OE mouse models, mass spectrometry, CRISPR mutagenesis, and Co-IP in a single rigorous study","pmids":["40856046"],"is_preprint":false},{"year":2023,"finding":"Loss of Gucy1a3 in mice worsens post-stroke recovery by increasing infarct volume and reducing microvessel density, VEGFA, and HIF-1α expression after permanent middle cerebral artery occlusion, placing GUCY1A3 upstream of the HIF-1α/VEGFA angiogenic signaling axis.","method":"Gucy1a3 knockout mouse model; TTC staining for infarct volume; CD31 immunohistochemistry for microvessel density; western blotting for VEGFA and HIF-1α","journal":"Journal of stroke and cerebrovascular diseases","confidence":"Medium","confidence_rationale":"Tier 2 — clean KO mouse with defined phenotypic readouts and pathway markers, single lab","pmids":["38064974"],"is_preprint":false},{"year":2023,"finding":"A GUCY1A3 missense variant (c.1778G>A) located in the catalytic domain of sGC is predicted to disrupt the 3D structure of that domain, leading to loss of enzymatic function; western blot confirmed reduced or absent protein expression in patient-derived cells, linking biallelic GUCY1A3 loss-of-function to moyamoya angiopathy via impaired NO-cGMP signaling.","method":"Exome sequencing; western blot of patient endothelial progenitor cells; protein 3D structure analysis","journal":"Human genomics","confidence":"Medium","confidence_rationale":"Tier 2-3 — western blot and structural modeling with patient-derived cells, single study","pmids":["36941667"],"is_preprint":false}],"current_model":"GUCY1A1/GUCY1A3 encodes the α1 subunit of soluble guanylate cyclase (sGC), which heterodimerizes with the β1 subunit and, upon nitric oxide stimulation, produces cGMP to activate PKG; PKG in turn phosphorylates LDHA (T95) to activate its moonlighting kinase function, which phosphorylates GPX4 (S131) to prevent its chaperone-mediated autophagic degradation and suppress endothelial ferroptosis, while the ZEB1 transcription factor regulates GUCY1A1 expression by binding its intronic regulatory region, and loss-of-function variants (missense or haploinsufficiency) reduce NO-stimulated cGMP production, impairing platelet inhibition, vascular smooth muscle relaxation, and angiogenesis, thereby predisposing to coronary artery disease, moyamoya angiopathy, hypertension, and microvascular reperfusion injury."},"narrative":{"teleology":[{"year":2004,"claim":"Establishing that GUCY1A1-driven cGMP production lies upstream of VEGF expression and angiogenesis resolved how sGC activity couples to tumor vascularization.","evidence":"Antisense knockdown in glioma cells with cGMP measurement, VEGF assays, HUVEC co-culture, and nude mouse tumor vascularization","pmids":["15201957"],"confidence":"Medium","gaps":["Mechanism linking cGMP to VEGF transcription not delineated","Antisense approach lacks genetic specificity of modern KO methods","Not replicated in non-tumor vascular beds"]},{"year":2014,"claim":"Demonstration that the α1-A680T variant produces a gain-of-function sGC with enhanced NO sensitivity established that GUCY1A1 activity is tunable in both directions and that increased function protects against pulmonary hypertension.","evidence":"Purified mutant protein enzymatic assay and reporter cell cGMP assay","pmids":["25373139"],"confidence":"Medium","gaps":["Single variant studied","No in vivo confirmation of hemodynamic protection by this allele alone","Structural basis for enhanced NO sensitivity not resolved"]},{"year":2016,"claim":"Functional characterization of multiple rare coding variants from MI patients demonstrated that loss-of-function mutations reduce NO-stimulated cGMP production while preserving β1 heterodimerization, and that pharmacological sGC stimulation can rescue activity, defining a therapeutic rationale.","evidence":"Co-immunoprecipitation for dimerization, cGMP radioimmunoassay in HEK293 cells, pharmacological rescue with BAY 41-2272","pmids":["27342234"],"confidence":"High","gaps":["Rescue not tested in vivo or in patient-derived cells","Platelet and vascular phenotypes not directly measured for each variant"]},{"year":2016,"claim":"Characterization of the C517Y missense variant as catalytically impaired directly linked a specific GUCY1A1 mutation to moyamoya disease and hypertension through blunted NO-cGMP signaling.","evidence":"Biochemical functional assay of mutant protein response to NO donor in vitro","pmids":["26777256"],"confidence":"Medium","gaps":["Single variant in single family","No vascular cell or animal model confirmation","Protein stability and expression levels not fully characterized"]},{"year":2017,"claim":"Identifying ZEB1 as a transcriptional regulator of GUCY1A1 via allele-specific binding at rs7692387 connected a common CAD-risk SNP to a molecular mechanism controlling sGC expression and downstream platelet and vascular smooth muscle function.","evidence":"Allele-specific ChIP, siRNA/overexpression of ZEB1, reporter assays, ex vivo platelet aggregation, VSMC migration assay","pmids":["28487391"],"confidence":"High","gaps":["Other transcription factors at this locus not excluded","In vivo confirmation of ZEB1 regulation of GUCY1A1 in arterial tissue lacking"]},{"year":2023,"claim":"Endothelial-specific Gucy1a1 knockout in mice revealed a PKG→LDHA(T95-P)→GPX4(S131-P) signaling cascade that suppresses ferroptosis by blocking chaperone-mediated autophagic degradation of GPX4, explaining how sGC-cGMP protects against microvascular no-reflow injury.","evidence":"EC-specific conditional KO and AAV overexpression in mice; mass spectrometry; CRISPR mutagenesis of phosphosites; Co-IP; CMA assay","pmids":["40856046"],"confidence":"High","gaps":["LDHA's moonlighting kinase activity awaits structural characterization","Relevance of this pathway in non-cardiac vascular beds not tested","Whether other PKG substrates contribute to anti-ferroptotic protection is unknown"]},{"year":2023,"claim":"Gucy1a3 knockout mice showed worsened stroke outcomes with reduced HIF-1α and VEGFA, positioning sGC upstream of the HIF-1α/VEGFA angiogenic axis in cerebral ischemia.","evidence":"KO mouse permanent MCAO model; TTC staining; CD31 immunohistochemistry; western blot for HIF-1α/VEGFA","pmids":["38064974"],"confidence":"Medium","gaps":["Direct cGMP-dependent mechanism linking sGC to HIF-1α stabilization unresolved","Single lab, single ischemia model","Endothelial versus neuronal contributions not dissected"]},{"year":2023,"claim":"A biallelic catalytic-domain missense variant confirmed by absent protein expression in patient cells solidified the genetic basis for moyamoya angiopathy as a loss-of-function GUCY1A1 disease.","evidence":"Exome sequencing, western blot of patient endothelial progenitor cells, 3D structural modeling","pmids":["36941667"],"confidence":"Medium","gaps":["No enzymatic activity assay of the mutant protein performed","Rescue experiment not attempted","Small number of patients studied"]},{"year":null,"claim":"It remains unknown how sGC-derived cGMP mechanistically stabilizes HIF-1α, whether the LDHA moonlighting kinase pathway operates in non-cardiac vascular beds, and what structural features govern the differential activity of disease-associated α1 variants.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural model of full-length α1β1 heterodimer with disease mutations mapped","cGMP-to-HIF-1α link not biochemically resolved","Tissue-specific requirements for sGC anti-ferroptotic signaling unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0009975","term_label":"cyclase activity","supporting_discovery_ids":[0,1,2]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[3]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[2,5]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,2,3,5]},{"term_id":"R-HSA-109582","term_label":"Hemostasis","supporting_discovery_ids":[3]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[5]}],"complexes":["soluble guanylate cyclase (α1β1 heterodimer)"],"partners":["GUCY1B3","ZEB1","PRKG1","LDHA","GPX4"],"other_free_text":[]},"mechanistic_narrative":"GUCY1A1 encodes the α1 subunit of soluble guanylate cyclase (sGC), which heterodimerizes with the β1 subunit and catalyzes cGMP production upon nitric oxide (NO) stimulation to mediate vascular smooth muscle relaxation, platelet inhibition, angiogenesis, and endothelial cell survival [PMID:27342234, PMID:28487391, PMID:40856046]. The transcription factor ZEB1 drives GUCY1A1 expression by binding an intronic regulatory element, and higher α1 levels enhance cGMP-mediated inhibition of platelet aggregation and vascular smooth muscle cell migration [PMID:28487391]. Downstream of sGC-generated cGMP, PKG phosphorylates LDHA at T95, activating a moonlighting kinase function that phosphorylates GPX4 at S131 to prevent its chaperone-mediated autophagic degradation, thereby suppressing endothelial ferroptosis and protecting against microvascular no-reflow injury [PMID:40856046]. Loss-of-function variants in GUCY1A1 cause blunted NO–cGMP signaling and are linked to moyamoya angiopathy, coronary artery disease, and hypertension [PMID:26777256, PMID:27342234, PMID:36941667]."},"prefetch_data":{"uniprot":{"accession":"Q02108","full_name":"Guanylate cyclase soluble subunit alpha-1","aliases":["Guanylate cyclase soluble subunit alpha-3","GCS-alpha-3","Soluble guanylate cyclase large subunit"],"length_aa":690,"mass_kda":77.5,"function":"","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q02108/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/GUCY1A1","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/GUCY1A1","total_profiled":1310},"omim":[{"mim_id":"615750","title":"MOYAMOYA DISEASE 6 WITH OR WITHOUT ACHALASIA; MYMY6","url":"https://www.omim.org/entry/615750"},{"mim_id":"139397","title":"GUANYLATE CYCLASE, SOLUBLE, BETA-1; GUCY1B1","url":"https://www.omim.org/entry/139397"},{"mim_id":"139396","title":"GUANYLATE CYCLASE, SOLUBLE, ALPHA-1; GUCY1A1","url":"https://www.omim.org/entry/139396"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/GUCY1A1"},"hgnc":{"alias_symbol":["GC-SA3"],"prev_symbol":["GUC1A3","GUCY1A3"]},"alphafold":{"accession":"Q02108","domains":[{"cath_id":"3.90.1520","chopping":"72-179_186-198_207-244","consensus_level":"high","plddt":81.0018,"start":72,"end":244},{"cath_id":"3.30.450.260","chopping":"280-355_363-404","consensus_level":"high","plddt":85.7872,"start":280,"end":404},{"cath_id":"3.30.70.1230","chopping":"470-661","consensus_level":"high","plddt":92.7219,"start":470,"end":661},{"cath_id":"1.20.5","chopping":"406-458","consensus_level":"medium","plddt":91.3215,"start":406,"end":458}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q02108","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q02108-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q02108-F1-predicted_aligned_error_v6.png","plddt_mean":77.44},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=GUCY1A1","jax_strain_url":"https://www.jax.org/strain/search?query=GUCY1A1"},"sequence":{"accession":"Q02108","fasta_url":"https://rest.uniprot.org/uniprotkb/Q02108.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q02108/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q02108"}},"corpus_meta":[{"pmid":"28487391","id":"PMC_28487391","title":"Functional Characterization of the GUCY1A3 Coronary Artery Disease Risk Locus.","date":"2017","source":"Circulation","url":"https://pubmed.ncbi.nlm.nih.gov/28487391","citation_count":81,"is_preprint":false},{"pmid":"34381413","id":"PMC_34381413","title":"RNF213 and GUCY1A3 in Moyamoya Disease: Key Regulators of Metabolism, Inflammation, and Vascular Stability.","date":"2021","source":"Frontiers in neurology","url":"https://pubmed.ncbi.nlm.nih.gov/34381413","citation_count":68,"is_preprint":false},{"pmid":"26777256","id":"PMC_26777256","title":"Disrupted nitric oxide signaling due to GUCY1A3 mutations increases risk for moyamoya disease, achalasia and hypertension.","date":"2016","source":"Clinical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/26777256","citation_count":63,"is_preprint":false},{"pmid":"15201957","id":"PMC_15201957","title":"Inhibition of angiogenesis in human glioma cell lines by antisense RNA from the soluble guanylate cyclase genes, GUCY1A3 and GUCY1B3.","date":"2004","source":"Oncology reports","url":"https://pubmed.ncbi.nlm.nih.gov/15201957","citation_count":32,"is_preprint":false},{"pmid":"31228190","id":"PMC_31228190","title":"Genetic variation at the coronary artery disease risk locus GUCY1A3 modifies cardiovascular disease prevention effects of aspirin.","date":"2019","source":"European heart journal","url":"https://pubmed.ncbi.nlm.nih.gov/31228190","citation_count":31,"is_preprint":false},{"pmid":"25373139","id":"PMC_25373139","title":"α1-A680T variant in GUCY1A3 as a candidate conferring protection from pulmonary hypertension among Kyrgyz highlanders.","date":"2014","source":"Circulation. Cardiovascular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/25373139","citation_count":23,"is_preprint":false},{"pmid":"30768153","id":"PMC_30768153","title":"Association of the coronary artery disease risk gene GUCY1A3 with ischaemic events after coronary intervention.","date":"2019","source":"Cardiovascular research","url":"https://pubmed.ncbi.nlm.nih.gov/30768153","citation_count":20,"is_preprint":false},{"pmid":"27342234","id":"PMC_27342234","title":"Stimulators of the soluble guanylyl cyclase: promising functional insights from rare coding atherosclerosis-related GUCY1A3 variants.","date":"2016","source":"Basic research in cardiology","url":"https://pubmed.ncbi.nlm.nih.gov/27342234","citation_count":20,"is_preprint":false},{"pmid":"36941667","id":"PMC_36941667","title":"Biallelic variants in NOS3 and GUCY1A3, the two major genes of the nitric oxide pathway, cause moyamoya cerebral angiopathy.","date":"2023","source":"Human genomics","url":"https://pubmed.ncbi.nlm.nih.gov/36941667","citation_count":8,"is_preprint":false},{"pmid":"40856046","id":"PMC_40856046","title":"GUCY1A1-LDHA Axis Suppresses Ferroptosis in Cardiac Ischemia-Reperfusion Injury.","date":"2025","source":"Circulation research","url":"https://pubmed.ncbi.nlm.nih.gov/40856046","citation_count":7,"is_preprint":false},{"pmid":"36289756","id":"PMC_36289756","title":"SELL and GUCY1A1 Gene Polymorphisms in Patients with Unstable Angina.","date":"2022","source":"Biomedicines","url":"https://pubmed.ncbi.nlm.nih.gov/36289756","citation_count":7,"is_preprint":false},{"pmid":"31883534","id":"PMC_31883534","title":"Associations between GUCY1A3 genetic polymorphisms and large artery atherosclerotic stroke risk in Chinese Han population: a case-control study.","date":"2019","source":"Lipids in health and disease","url":"https://pubmed.ncbi.nlm.nih.gov/31883534","citation_count":4,"is_preprint":false},{"pmid":"38064974","id":"PMC_38064974","title":"Loss of Gucy1a3 causes poor post-stroke recovery by reducing angiogenesis via the HIF-1α/VEGFA signaling pathway in mice.","date":"2023","source":"Journal of stroke and cerebrovascular diseases : the official journal of National Stroke Association","url":"https://pubmed.ncbi.nlm.nih.gov/38064974","citation_count":2,"is_preprint":false},{"pmid":"41761525","id":"PMC_41761525","title":"[Polymorphism rs7692387 of GUCY1A1 as a genetic marker for peripheral artery disease in cigarette smokers].","date":"2022","source":"Angiologiia i sosudistaia khirurgiia = Angiology and vascular surgery","url":"https://pubmed.ncbi.nlm.nih.gov/41761525","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.08.22.24312458","title":"Triangulating Evidence for Antihypertensive Drug Class Efficacy on Cardiovascular and Metabolic Outcomes Using Mendelian Randomisation and Colocalisation","date":"2024-08-23","source":"bioRxiv","url":"https://doi.org/10.1101/2024.08.22.24312458","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11109,"output_tokens":2214,"usd":0.033268},"stage2":{"model":"claude-opus-4-6","input_tokens":5524,"output_tokens":2284,"usd":0.12708},"total_usd":0.160348,"stage1_batch_id":"msgbatch_01DzrsgVgYngY1hvM3DU1yF2","stage2_batch_id":"msgbatch_01KA9b7iRzKFdhoBUT7vCvSe","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2016,\n      \"finding\": \"GUCY1A3 encodes the α1 subunit of soluble guanylate cyclase (sGC); a missense variant (Cys517Tyr) in GUCY1A3 produces a mutant α1 protein with significantly blunted cGMP signaling response upon nitric oxide (NO) exposure, establishing loss-of-function as the mechanism linking GUCY1A3 mutations to moyamoya disease and hypertension.\",\n      \"method\": \"Biochemical functional assay of mutant protein signaling response to NO donor in vitro\",\n      \"journal\": \"Clinical genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct functional assay of mutant protein, single lab/study\",\n      \"pmids\": [\"26777256\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"A missense variant α1-A680T in GUCY1A3 results in higher cGMP production in reporter cells and enhanced sensitivity to nitric oxide in purified protein in vitro, conferring a gain-of-function phenotype protective against high-altitude pulmonary hypertension.\",\n      \"method\": \"Reporter cell cGMP assay and in vitro enzymatic assay of purified α1-A680T sGC\",\n      \"journal\": \"Circulation. Cardiovascular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — in vitro enzymatic assay with purified protein and cell-based assay, single study\",\n      \"pmids\": [\"25373139\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Rare coding GUCY1A3 variants found in MI patients all dimerize with the β1 subunit (co-immunoprecipitation); five variants display decreased cGMP production upon NO stimulation, and this reduced activity is rescued by the sGC stimulator BAY 41-2272 in vitro.\",\n      \"method\": \"Co-immunoprecipitation for dimerization; cGMP radioimmunoassay after NO donor stimulation in HEK293 cells; pharmacological rescue with BAY 41-2272\",\n      \"journal\": \"Basic research in cardiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods (Co-IP, enzymatic assay, pharmacological rescue) in single study\",\n      \"pmids\": [\"27342234\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"The transcription factor ZEB1 binds preferentially to the non-risk allele of rs7692387 (intronic site), driving higher GUCY1A3 promoter activity and expression; ZEB1 knockdown reduces non-risk allele promoter activity and endogenous GUCY1A3 expression. Higher sGC (α1) levels lead to enhanced cGMP-mediated inhibition of ADP-induced platelet aggregation and reduced vascular smooth muscle cell migration upon pharmacological sGC stimulation.\",\n      \"method\": \"Allele-specific ChIP; siRNA knockdown and overexpression of ZEB1; reporter gene assays; platelet aggregation assay ex vivo; VSMC migration assay\",\n      \"journal\": \"Circulation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (ChIP, siRNA, reporter assay, functional cell assays), replicated in human ex vivo samples\",\n      \"pmids\": [\"28487391\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Antisense knockdown of GUCY1A3 (or GUCY1B3) in glioma cell lines markedly reduced cGMP content and VEGF expression, inhibited HUVEC growth in vitro, and suppressed subcutaneous tumor vascularization in vivo, establishing that GUCY1A3-driven cGMP production is an upstream mediator of VEGF expression and angiogenesis in glioma.\",\n      \"method\": \"Antisense RNA transfection; cGMP measurement; VEGF expression assay; HUVEC co-culture angiogenesis assay; nude mouse tumor model with vascular index quantification\",\n      \"journal\": \"Oncology reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — loss-of-function with multiple phenotypic readouts in vitro and in vivo, single lab\",\n      \"pmids\": [\"15201957\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Endothelial cell-specific knockout of GUCY1A1 in mice increases microvascular no-reflow area, infarction size, and cardiac dysfunction after ischemia-reperfusion injury; mechanistically, PKG (downstream of sGC-cGMP) phosphorylates LDHA at threonine 95, activating LDHA's moonlighting kinase function to phosphorylate GPX4 at serine 131, reducing chaperone-mediated autophagy-dependent GPX4 degradation and thereby suppressing ferroptosis in endothelial cells.\",\n      \"method\": \"EC-specific conditional knockout and AAV-mediated overexpression in mice; mass spectrometry identification of phosphorylation sites; CRISPR-Cas9 mutagenesis of phosphorylation sites; co-immunoprecipitation for protein interactions; chaperone-mediated autophagy assay\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods including KO/OE mouse models, mass spectrometry, CRISPR mutagenesis, and Co-IP in a single rigorous study\",\n      \"pmids\": [\"40856046\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Loss of Gucy1a3 in mice worsens post-stroke recovery by increasing infarct volume and reducing microvessel density, VEGFA, and HIF-1α expression after permanent middle cerebral artery occlusion, placing GUCY1A3 upstream of the HIF-1α/VEGFA angiogenic signaling axis.\",\n      \"method\": \"Gucy1a3 knockout mouse model; TTC staining for infarct volume; CD31 immunohistochemistry for microvessel density; western blotting for VEGFA and HIF-1α\",\n      \"journal\": \"Journal of stroke and cerebrovascular diseases\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean KO mouse with defined phenotypic readouts and pathway markers, single lab\",\n      \"pmids\": [\"38064974\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"A GUCY1A3 missense variant (c.1778G>A) located in the catalytic domain of sGC is predicted to disrupt the 3D structure of that domain, leading to loss of enzymatic function; western blot confirmed reduced or absent protein expression in patient-derived cells, linking biallelic GUCY1A3 loss-of-function to moyamoya angiopathy via impaired NO-cGMP signaling.\",\n      \"method\": \"Exome sequencing; western blot of patient endothelial progenitor cells; protein 3D structure analysis\",\n      \"journal\": \"Human genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — western blot and structural modeling with patient-derived cells, single study\",\n      \"pmids\": [\"36941667\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"GUCY1A1/GUCY1A3 encodes the α1 subunit of soluble guanylate cyclase (sGC), which heterodimerizes with the β1 subunit and, upon nitric oxide stimulation, produces cGMP to activate PKG; PKG in turn phosphorylates LDHA (T95) to activate its moonlighting kinase function, which phosphorylates GPX4 (S131) to prevent its chaperone-mediated autophagic degradation and suppress endothelial ferroptosis, while the ZEB1 transcription factor regulates GUCY1A1 expression by binding its intronic regulatory region, and loss-of-function variants (missense or haploinsufficiency) reduce NO-stimulated cGMP production, impairing platelet inhibition, vascular smooth muscle relaxation, and angiogenesis, thereby predisposing to coronary artery disease, moyamoya angiopathy, hypertension, and microvascular reperfusion injury.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"GUCY1A1 encodes the α1 subunit of soluble guanylate cyclase (sGC), which heterodimerizes with the β1 subunit and catalyzes cGMP production upon nitric oxide (NO) stimulation to mediate vascular smooth muscle relaxation, platelet inhibition, angiogenesis, and endothelial cell survival [PMID:27342234, PMID:28487391, PMID:40856046]. The transcription factor ZEB1 drives GUCY1A1 expression by binding an intronic regulatory element, and higher α1 levels enhance cGMP-mediated inhibition of platelet aggregation and vascular smooth muscle cell migration [PMID:28487391]. Downstream of sGC-generated cGMP, PKG phosphorylates LDHA at T95, activating a moonlighting kinase function that phosphorylates GPX4 at S131 to prevent its chaperone-mediated autophagic degradation, thereby suppressing endothelial ferroptosis and protecting against microvascular no-reflow injury [PMID:40856046]. Loss-of-function variants in GUCY1A1 cause blunted NO–cGMP signaling and are linked to moyamoya angiopathy, coronary artery disease, and hypertension [PMID:26777256, PMID:27342234, PMID:36941667].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Establishing that GUCY1A1-driven cGMP production lies upstream of VEGF expression and angiogenesis resolved how sGC activity couples to tumor vascularization.\",\n      \"evidence\": \"Antisense knockdown in glioma cells with cGMP measurement, VEGF assays, HUVEC co-culture, and nude mouse tumor vascularization\",\n      \"pmids\": [\"15201957\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking cGMP to VEGF transcription not delineated\", \"Antisense approach lacks genetic specificity of modern KO methods\", \"Not replicated in non-tumor vascular beds\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Demonstration that the α1-A680T variant produces a gain-of-function sGC with enhanced NO sensitivity established that GUCY1A1 activity is tunable in both directions and that increased function protects against pulmonary hypertension.\",\n      \"evidence\": \"Purified mutant protein enzymatic assay and reporter cell cGMP assay\",\n      \"pmids\": [\"25373139\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single variant studied\", \"No in vivo confirmation of hemodynamic protection by this allele alone\", \"Structural basis for enhanced NO sensitivity not resolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Functional characterization of multiple rare coding variants from MI patients demonstrated that loss-of-function mutations reduce NO-stimulated cGMP production while preserving β1 heterodimerization, and that pharmacological sGC stimulation can rescue activity, defining a therapeutic rationale.\",\n      \"evidence\": \"Co-immunoprecipitation for dimerization, cGMP radioimmunoassay in HEK293 cells, pharmacological rescue with BAY 41-2272\",\n      \"pmids\": [\"27342234\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Rescue not tested in vivo or in patient-derived cells\", \"Platelet and vascular phenotypes not directly measured for each variant\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Characterization of the C517Y missense variant as catalytically impaired directly linked a specific GUCY1A1 mutation to moyamoya disease and hypertension through blunted NO-cGMP signaling.\",\n      \"evidence\": \"Biochemical functional assay of mutant protein response to NO donor in vitro\",\n      \"pmids\": [\"26777256\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single variant in single family\", \"No vascular cell or animal model confirmation\", \"Protein stability and expression levels not fully characterized\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identifying ZEB1 as a transcriptional regulator of GUCY1A1 via allele-specific binding at rs7692387 connected a common CAD-risk SNP to a molecular mechanism controlling sGC expression and downstream platelet and vascular smooth muscle function.\",\n      \"evidence\": \"Allele-specific ChIP, siRNA/overexpression of ZEB1, reporter assays, ex vivo platelet aggregation, VSMC migration assay\",\n      \"pmids\": [\"28487391\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Other transcription factors at this locus not excluded\", \"In vivo confirmation of ZEB1 regulation of GUCY1A1 in arterial tissue lacking\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Endothelial-specific Gucy1a1 knockout in mice revealed a PKG→LDHA(T95-P)→GPX4(S131-P) signaling cascade that suppresses ferroptosis by blocking chaperone-mediated autophagic degradation of GPX4, explaining how sGC-cGMP protects against microvascular no-reflow injury.\",\n      \"evidence\": \"EC-specific conditional KO and AAV overexpression in mice; mass spectrometry; CRISPR mutagenesis of phosphosites; Co-IP; CMA assay\",\n      \"pmids\": [\"40856046\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"LDHA's moonlighting kinase activity awaits structural characterization\", \"Relevance of this pathway in non-cardiac vascular beds not tested\", \"Whether other PKG substrates contribute to anti-ferroptotic protection is unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Gucy1a3 knockout mice showed worsened stroke outcomes with reduced HIF-1α and VEGFA, positioning sGC upstream of the HIF-1α/VEGFA angiogenic axis in cerebral ischemia.\",\n      \"evidence\": \"KO mouse permanent MCAO model; TTC staining; CD31 immunohistochemistry; western blot for HIF-1α/VEGFA\",\n      \"pmids\": [\"38064974\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct cGMP-dependent mechanism linking sGC to HIF-1α stabilization unresolved\", \"Single lab, single ischemia model\", \"Endothelial versus neuronal contributions not dissected\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"A biallelic catalytic-domain missense variant confirmed by absent protein expression in patient cells solidified the genetic basis for moyamoya angiopathy as a loss-of-function GUCY1A1 disease.\",\n      \"evidence\": \"Exome sequencing, western blot of patient endothelial progenitor cells, 3D structural modeling\",\n      \"pmids\": [\"36941667\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No enzymatic activity assay of the mutant protein performed\", \"Rescue experiment not attempted\", \"Small number of patients studied\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unknown how sGC-derived cGMP mechanistically stabilizes HIF-1α, whether the LDHA moonlighting kinase pathway operates in non-cardiac vascular beds, and what structural features govern the differential activity of disease-associated α1 variants.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model of full-length α1β1 heterodimer with disease mutations mapped\", \"cGMP-to-HIF-1α link not biochemically resolved\", \"Tissue-specific requirements for sGC anti-ferroptotic signaling unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0009975\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"GO:0016849\", \"supporting_discovery_ids\": []},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2, 5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 2, 3, 5]},\n      {\"term_id\": \"R-HSA-109582\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"complexes\": [\n      \"soluble guanylate cyclase (α1β1 heterodimer)\"\n    ],\n    \"partners\": [\n      \"GUCY1B3\",\n      \"ZEB1\",\n      \"PRKG1\",\n      \"LDHA\",\n      \"GPX4\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}