{"gene":"ASB4","run_date":"2026-06-09T22:02:44","timeline":{"discoveries":[{"year":2007,"finding":"ASB4 is a substrate for FIH (factor inhibiting HIF1α)-mediated asparagine hydroxylation via an oxygen-dependent mechanism; ASB4 interacts with FIH and is hydroxylated by FIH in normoxia, which is postulated to promote substrate binding and degradation.","method":"Co-immunoprecipitation, hydroxylation assay, overexpression in ES cells with oxygen-dependent differentiation readout","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal interaction demonstrated by Co-IP, oxygen-dependent functional assay, single lab with two orthogonal methods","pmids":["17636018"],"is_preprint":false},{"year":2007,"finding":"ASB4 functions as the substrate recognition subunit of an elongin B/elongin C/cullin/Roc E3 ubiquitin ligase complex, mediating ubiquitination and proteasomal degradation of substrate proteins; overexpression of ASB4 in embryonic stem cells promotes differentiation into the vascular lineage.","method":"Biochemical characterization of SOCS box complex; ES cell overexpression with vascular differentiation assay","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — complex membership inferred from SOCS box domain characterization plus functional overexpression assay, single lab","pmids":["17636018","11111040"],"is_preprint":false},{"year":2014,"finding":"ASB4 ubiquitinates and promotes proteasome-dependent degradation of the transcriptional regulator ID2 in trophoblast cells, thereby promoting trophoblast differentiation and vascular patterning in the placenta; co-transfection of a degradation-resistant ID2 mutant with ASB4 inhibits both differentiation and functional vascular responses.","method":"Co-immunoprecipitation, ubiquitination assay, proteasome inhibitor treatment, degradation-resistant ID2 mutant rescue, endothelial co-culture functional assay, Asb4 knockout mouse placental phenotyping","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods including ubiquitination assay, proteasome-dependent degradation, dominant-negative rescue, in vivo KO phenotype, all in single study","pmids":["24586788"],"is_preprint":false},{"year":2007,"finding":"ASB4 (Asb-4) interacts with GPS1 (CSN1) via its ankyrin repeat domain (independent of the SOCS box) and reduces GPS1 protein levels; co-expression of ASB4 with GPS1 inhibits c-Jun NH2-terminal kinase (JNK) activity and reduces insulin-stimulated IRS-1 serine 307 phosphorylation.","method":"Yeast two-hybrid screening, co-immunoprecipitation in vitro and in HEK293 cells, SOCS box deletion mutant, JNK activity assay, IRS-1 phosphorylation assay","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid plus reciprocal Co-IP plus deletion mutagenesis plus kinase activity assay, single lab","pmids":["17276034"],"is_preprint":false},{"year":2011,"finding":"ASB4 co-localizes with IRS4 in hypothalamic POMC and NPY neurons, physically interacts with IRS4 (confirmed by Co-IP in cell lines and rat hypothalamic extracts), ubiquitinates IRS4 in a SOCS box-dependent manner, promotes IRS4 proteasomal degradation, and reduces both basal and insulin-stimulated AKT (Thr308) phosphorylation.","method":"In situ hybridization co-localization, co-immunoprecipitation (heterologous cells and endogenous hypothalamic extracts), SOCS box deletion mutant, ubiquitination assay, AKT phosphorylation assay","journal":"BMC neuroscience","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods: co-IP in native tissue, ubiquitination assay, mutagenesis of SOCS box, downstream signaling readout, single lab but rigorous","pmids":["21955513"],"is_preprint":false},{"year":2009,"finding":"Overexpression of ASB4 specifically in POMC neurons of the arcuate nucleus increases food intake, reduces fat mass, increases lean mass, raises metabolic rate (O2 consumption and CO2 production), increases locomotor activity, and elevates POMC mRNA; ASB4 expression in the hypothalamus is regulated by insulin (paraventricular nucleus) and leptin (paraventricular nucleus and arcuate nucleus).","method":"Transgenic mouse model (POMC-Asb4), metabolic cage analysis, intracerebroventricular insulin injection, intraperitoneal leptin injection, quantitative gene expression","journal":"Endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean transgenic gain-of-function with defined metabolic phenotype, multiple physiological readouts, single lab","pmids":["19934378"],"is_preprint":false},{"year":2022,"finding":"ASB4 acts downstream of AgRP in the hypothalamus to regulate satiety and glucose homeostasis: hypothalamic Asb4 expression is suppressed by fasting in an AgRP-dependent manner; acute Asb4 knockdown causes hyperphagia via increased meal size; Asb4-deficient mice are resistant to calcitonin-induced meal termination and show reduced Calcr (calcitonin receptor) expression in neurons; POMC neuron-specific Asb4 deletion causes glucose intolerance independent of obesity.","method":"AgRP-knockout mice, acute siRNA knockdown, Asb4 global knockout, POMC-specific conditional knockout, calcitonin pharmacological challenge, qPCR for Calcr, metabolic and glucose tolerance testing","journal":"Science signaling","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple genetic models (global KO, POMC-specific KO, AgRP-KO epistasis), pharmacological challenge, defined molecular and behavioral phenotypes, single lab but multiple orthogonal approaches","pmids":["35536884"],"is_preprint":false},{"year":2023,"finding":"In the absence of ASB4, insulin (but not leptin) elevates ID2 protein levels post-transcriptionally in trophoblasts, implicating hyperinsulinemia in perturbing ASB4-mediated ID2 degradation and contributing to enhanced preeclampsia pathology.","method":"Asb4-null mice on high-fat diet, placental ID2 protein and mRNA quantification, insulin/leptin treatment of HTR8/SVneo human trophoblast cells","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo KO plus cell-based hormone treatment, protein vs. transcript comparison, single lab","pmids":["36768469"],"is_preprint":false},{"year":2002,"finding":"Asb4 is an imprinted gene showing differential expression between parthenogenetic and androgenetic mouse embryos, confirmed in normal diploid embryos from reciprocal F1 crosses.","method":"RIKEN cDNA microarray screen, reciprocal F1 cross validation by expression analysis","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — microarray with genetic cross validation, replicated in multiple cross directions but no mechanistic follow-up on imprinting mechanism","pmids":["11820791"],"is_preprint":false},{"year":2014,"finding":"ASB4 expression promotes migration and invasion of hepatocellular carcinoma (HCC) cells; suppression of ASB4 in HCC cell lines (PLC, MHCC97-L) reduces migration and invasion, while ectopic ASB4 expression in Hep3B cells enhances migration; ASB4 mRNA levels are negatively regulated by miR-200a through a validated binding site in the ASB4 3' UTR.","method":"siRNA knockdown and overexpression in HCC cell lines, migration/invasion assays, dual luciferase reporter assay for miR-200a 3' UTR binding","journal":"Bioscience trends","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — loss- and gain-of-function migration assays plus luciferase reporter for miRNA regulation, single lab","pmids":["24815387"],"is_preprint":false}],"current_model":"ASB4 is a SOCS box-containing E3 ubiquitin ligase substrate-recognition subunit that assembles with elongin B/C/cullin/Roc to ubiquitinate and degrade specific substrates—including ID2 (in trophoblasts promoting placental vascular differentiation), IRS4 (in hypothalamic neurons modulating insulin signaling), and GPS1/CSN1 (suppressing JNK activity)—and whose activity is itself regulated by oxygen-dependent FIH-mediated hydroxylation; in the hypothalamus ASB4 acts downstream of AgRP to regulate satiety via calcitonin receptor expression and controls glucose homeostasis specifically in POMC neurons."},"narrative":{"mechanistic_narrative":"ASB4 is the substrate-recognition subunit of an elongin B/elongin C/cullin/Roc E3 ubiquitin ligase complex that targets specific proteins for ubiquitination and proteasomal degradation, coupling oxygen sensing and metabolic signaling to differentiation and energy homeostasis [PMID:17636018, PMID:11111040]. Its ligase activity is regulated by oxygen tension: FIH hydroxylates an asparagine residue in ASB4 in normoxia, an oxygen-dependent modification linked to substrate binding and degradation [PMID:17636018]. Through its SOCS box, ASB4 ubiquitinates and degrades the transcriptional regulator ID2 in trophoblasts, driving trophoblast differentiation and placental vascular patterning, and a degradation-resistant ID2 mutant blocks both responses [PMID:24586788]; loss of ASB4 allows insulin to elevate ID2 post-transcriptionally, linking the pathway to preeclampsia pathology [PMID:36768469]. In hypothalamic POMC and NPY neurons, ASB4 binds and ubiquitinates IRS4 in a SOCS box-dependent manner to dampen insulin/AKT signaling [PMID:21955513], and acts downstream of AgRP to regulate satiety—being suppressed by fasting, required for calcitonin-induced meal termination via Calcr expression, and controlling glucose homeostasis in POMC neurons [PMID:35536884]. Separately, ASB4 engages GPS1/CSN1 through its ankyrin-repeat domain, independent of the SOCS box, to suppress JNK activity and reduce IRS-1 serine phosphorylation [PMID:17276034].","teleology":[{"year":2002,"claim":"Established Asb4 as an imprinted gene, the first indication that its expression is under parent-of-origin genomic control.","evidence":"RIKEN cDNA microarray of parthenogenetic vs androgenetic embryos with reciprocal F1 cross validation","pmids":["11820791"],"confidence":"Medium","gaps":["No mechanism of imprinting (DMR, methylation) characterized","No link drawn between imprinting and ASB4 protein function"]},{"year":2007,"claim":"Defined the core molecular identity of ASB4 as a SOCS-box E3 ubiquitin ligase substrate-recognition subunit and tied its activity to oxygen via FIH hydroxylation, answering what biochemical machine ASB4 belongs to.","evidence":"SOCS box complex biochemistry, Co-IP and hydroxylation assay with FIH, ES cell overexpression with oxygen-dependent vascular differentiation readout","pmids":["17636018","11111040"],"confidence":"Medium","gaps":["Endogenous substrate(s) in ES/vascular context not identified","Functional consequence of hydroxylation on substrate degradation not directly demonstrated"]},{"year":2007,"claim":"Revealed a SOCS-box-independent activity: ASB4 binds GPS1/CSN1 through its ankyrin repeats to suppress JNK and IRS-1 serine phosphorylation, expanding its role beyond canonical ligase function.","evidence":"Yeast two-hybrid, reciprocal Co-IP, SOCS box deletion mutant, JNK and IRS-1 phosphorylation assays in HEK293 cells","pmids":["17276034"],"confidence":"Medium","gaps":["Whether GPS1 reduction is ubiquitin-dependent not resolved","In vivo relevance of the ASB4-GPS1-JNK axis untested"]},{"year":2009,"claim":"Connected ASB4 to whole-animal energy balance by showing POMC-neuron overexpression alters food intake, body composition and metabolic rate, and that its hypothalamic expression responds to insulin and leptin.","evidence":"POMC-Asb4 transgenic mice, metabolic cage analysis, ICV insulin and IP leptin challenge, gene expression","pmids":["19934378"],"confidence":"Medium","gaps":["Molecular substrate mediating the metabolic phenotype not identified in this study","Gain-of-function only; loss-of-function not yet tested"]},{"year":2011,"claim":"Identified IRS4 as a neuronal substrate, providing a molecular substrate that links ASB4 ligase activity to attenuated insulin/AKT signaling in hypothalamic neurons.","evidence":"In situ co-localization, Co-IP in cells and endogenous hypothalamic extracts, SOCS box deletion mutant, ubiquitination and AKT phosphorylation assays","pmids":["21955513"],"confidence":"High","gaps":["In vivo requirement of IRS4 degradation for the metabolic phenotype not established","Relationship between IRS4 and downstream feeding circuits unresolved"]},{"year":2014,"claim":"Established ID2 as a physiological substrate in trophoblasts and connected ASB4-mediated ID2 degradation to placental differentiation and vascular patterning, with in vivo KO validation.","evidence":"Co-IP, ubiquitination assay, proteasome inhibition, degradation-resistant ID2 mutant rescue, endothelial co-culture, Asb4 knockout placental phenotyping","pmids":["24586788"],"confidence":"High","gaps":["Whether FIH hydroxylation gates ID2 degradation not tested here","Upstream signals controlling ASB4 in trophoblasts not defined"]},{"year":2014,"claim":"Implicated ASB4 in hepatocellular carcinoma cell motility and showed its mRNA is repressed by miR-200a, indicating a regulatory layer controlling ASB4 abundance.","evidence":"siRNA knockdown and overexpression in HCC cell lines, migration/invasion assays, dual luciferase reporter for miR-200a 3'UTR binding","pmids":["24815387"],"confidence":"Medium","gaps":["Ligase substrate driving migration not identified","No in vivo tumor model"]},{"year":2022,"claim":"Placed ASB4 in a defined feeding circuit: it acts downstream of AgRP, is fasting-suppressed, mediates calcitonin-induced satiety via Calcr, and controls glucose homeostasis specifically in POMC neurons.","evidence":"AgRP-KO epistasis, acute siRNA knockdown, global and POMC-specific Asb4 KO, calcitonin challenge, qPCR for Calcr, glucose tolerance testing","pmids":["35536884"],"confidence":"High","gaps":["Direct ligase substrate controlling Calcr expression not identified","Mechanism coupling ASB4 loss to glucose intolerance unresolved"]},{"year":2023,"claim":"Linked hyperinsulinemia to disease by showing that in ASB4 absence insulin elevates ID2 post-transcriptionally in trophoblasts, connecting failed ID2 degradation to preeclampsia pathology.","evidence":"Asb4-null mice on high-fat diet, placental ID2 protein vs mRNA, insulin/leptin treatment of HTR8/SVneo trophoblasts","pmids":["36768469"],"confidence":"Medium","gaps":["Causal chain from insulin to ASB4 activity not mechanistically dissected","Direct demonstration that ID2 stabilization drives the phenotype absent"]},{"year":null,"claim":"How oxygen-dependent FIH hydroxylation, substrate selection (ID2, IRS4, GPS1), and tissue context are integrated to direct ASB4 between vascular/placental differentiation and hypothalamic energy homeostasis remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of substrate recognition","Hydroxylation-substrate degradation coupling not directly demonstrated for any substrate","No unified in vivo test linking ligase activity to each phenotype"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,2,4]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[1,2,4]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,2,4]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[3,4]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,2,4]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,4,6]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[1,2]}],"complexes":["elongin B/elongin C/cullin/Roc E3 ubiquitin ligase"],"partners":["FIH","ID2","IRS4","GPS1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9Y574","full_name":"Ankyrin repeat and SOCS box protein 4","aliases":[],"length_aa":426,"mass_kda":48.2,"function":"Probable substrate-recognition component of a SCF-like ECS (Elongin-Cullin-SOCS-box protein) E3 ubiquitin-protein ligase complex which mediates the ubiquitination and subsequent proteasomal degradation of target proteins. Promotes differentiation and maturation of the vascular lineage by an oxygen-dependent mechanism (By similarity). Also acts as a negative regulator of GPS1, a component of the COP9 signalosome (CSN) multiprotein complex, thereby inhibiting the serine phosphorylation of IRS1 (By similarity). Regulates IRS4 levels by directing its degradation via ubiquitination and thereby decreases the downstream signal of IRS4 (PubMed:21955513). Plays a critical role during early vascular development and proper placentation. Mechanistically, negatively regulates the transcriptional regulator inhibitor of DNA binding 2/ID2 expression through polyubiquitination and proteasome dependent degradation (PubMed:24586788)","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q9Y574/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ASB4","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/ASB4","total_profiled":1310},"omim":[{"mim_id":"605761","title":"ANKYRIN REPEAT- AND SOCS BOX-CONTAINING PROTEIN 4; ASB4","url":"https://www.omim.org/entry/605761"},{"mim_id":"300904","title":"INSULIN RECEPTOR SUBSTRATE 4; IRS4","url":"https://www.omim.org/entry/300904"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"adrenal gland","ntpm":44.5},{"tissue":"skeletal muscle","ntpm":32.0}],"url":"https://www.proteinatlas.org/search/ASB4"},"hgnc":{"alias_symbol":["ASB-4"],"prev_symbol":[]},"alphafold":{"accession":"Q9Y574","domains":[{"cath_id":"1.25.40.20","chopping":"22-56_74-136","consensus_level":"medium","plddt":95.9036,"start":22,"end":136},{"cath_id":"1.25.40.20","chopping":"174-278","consensus_level":"medium","plddt":97.6974,"start":174,"end":278},{"cath_id":"-","chopping":"297-426","consensus_level":"medium","plddt":93.9943,"start":297,"end":426}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y574","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y574-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y574-F1-predicted_aligned_error_v6.png","plddt_mean":92.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ASB4","jax_strain_url":"https://www.jax.org/strain/search?query=ASB4"},"sequence":{"accession":"Q9Y574","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9Y574.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9Y574/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y574"}},"corpus_meta":[{"pmid":"11820791","id":"PMC_11820791","title":"Asb4, Ata3, and Dcn are novel imprinted genes identified by high-throughput screening using RIKEN cDNA microarray.","date":"2002","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/11820791","citation_count":116,"is_preprint":false},{"pmid":"17636018","id":"PMC_17636018","title":"ASB4 is a hydroxylation substrate of FIH and promotes vascular differentiation via an oxygen-dependent mechanism.","date":"2007","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/17636018","citation_count":83,"is_preprint":false},{"pmid":"11111040","id":"PMC_11111040","title":"Cloning and characterization of the genes encoding the ankyrin repeat and SOCS box-containing proteins Asb-1, Asb-2, Asb-3 and Asb-4.","date":"2000","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/11111040","citation_count":48,"is_preprint":false},{"pmid":"24586788","id":"PMC_24586788","title":"The ubiquitin ligase ASB4 promotes trophoblast differentiation through the degradation of ID2.","date":"2014","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/24586788","citation_count":42,"is_preprint":false},{"pmid":"29371260","id":"PMC_29371260","title":"The Antigen ASB4 on Cancer Stem Cells Serves as a Target for CTL Immunotherapy of Colorectal Cancer.","date":"2018","source":"Cancer immunology research","url":"https://pubmed.ncbi.nlm.nih.gov/29371260","citation_count":39,"is_preprint":false},{"pmid":"17276034","id":"PMC_17276034","title":"Ankyrin repeat and SOCS box containing protein 4 (Asb-4) interacts with GPS1 (CSN1) and inhibits c-Jun NH2-terminal kinase activity.","date":"2007","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/17276034","citation_count":24,"is_preprint":false},{"pmid":"21955513","id":"PMC_21955513","title":"Ankyrin repeat and SOCS box containing protein 4 (Asb-4) colocalizes with insulin receptor substrate 4 (IRS4) in the hypothalamic neurons and mediates IRS4 degradation.","date":"2011","source":"BMC neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/21955513","citation_count":24,"is_preprint":false},{"pmid":"19934378","id":"PMC_19934378","title":"Expression of ankyrin repeat and suppressor of cytokine signaling box protein 4 (Asb-4) in proopiomelanocortin neurons of the arcuate nucleus of mice produces a hyperphagic, lean phenotype.","date":"2009","source":"Endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/19934378","citation_count":16,"is_preprint":false},{"pmid":"24815387","id":"PMC_24815387","title":"Expression of ankyrin repeat and SOCS box containing 4 (ASB4) confers migration and invasion properties of hepatocellular carcinoma cells.","date":"2014","source":"Bioscience trends","url":"https://pubmed.ncbi.nlm.nih.gov/24815387","citation_count":15,"is_preprint":false},{"pmid":"35536884","id":"PMC_35536884","title":"ASB4 modulates central melanocortinergic neurons and calcitonin signaling to control satiety and glucose homeostasis.","date":"2022","source":"Science signaling","url":"https://pubmed.ncbi.nlm.nih.gov/35536884","citation_count":12,"is_preprint":false},{"pmid":"18414003","id":"PMC_18414003","title":"Stage-specific expression of ankyrin and SOCS box protein-4 (Asb-4) during spermatogenesis.","date":"2008","source":"Molecules and cells","url":"https://pubmed.ncbi.nlm.nih.gov/18414003","citation_count":8,"is_preprint":false},{"pmid":"36768469","id":"PMC_36768469","title":"Insulin Elevates ID2 Expression in Trophoblasts and Aggravates Preeclampsia in Obese ASB4-Null Mice.","date":"2023","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/36768469","citation_count":5,"is_preprint":false},{"pmid":"39201703","id":"PMC_39201703","title":"The Roles of Obesity and ASB4 in Preeclampsia Pathogenesis.","date":"2024","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/39201703","citation_count":3,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8637,"output_tokens":2647,"usd":0.032808,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9979,"output_tokens":3393,"usd":0.06736,"stage2_stop_reason":"end_turn"},"total_usd":0.100168,"stage1_batch_id":"msgbatch_0179Z2pxRYBsXi2xADneafRw","stage2_batch_id":"msgbatch_01LaY9ZaVdgjWArcZo9eqQE5","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2007,\n      \"finding\": \"ASB4 is a substrate for FIH (factor inhibiting HIF1α)-mediated asparagine hydroxylation via an oxygen-dependent mechanism; ASB4 interacts with FIH and is hydroxylated by FIH in normoxia, which is postulated to promote substrate binding and degradation.\",\n      \"method\": \"Co-immunoprecipitation, hydroxylation assay, overexpression in ES cells with oxygen-dependent differentiation readout\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal interaction demonstrated by Co-IP, oxygen-dependent functional assay, single lab with two orthogonal methods\",\n      \"pmids\": [\"17636018\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"ASB4 functions as the substrate recognition subunit of an elongin B/elongin C/cullin/Roc E3 ubiquitin ligase complex, mediating ubiquitination and proteasomal degradation of substrate proteins; overexpression of ASB4 in embryonic stem cells promotes differentiation into the vascular lineage.\",\n      \"method\": \"Biochemical characterization of SOCS box complex; ES cell overexpression with vascular differentiation assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — complex membership inferred from SOCS box domain characterization plus functional overexpression assay, single lab\",\n      \"pmids\": [\"17636018\", \"11111040\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ASB4 ubiquitinates and promotes proteasome-dependent degradation of the transcriptional regulator ID2 in trophoblast cells, thereby promoting trophoblast differentiation and vascular patterning in the placenta; co-transfection of a degradation-resistant ID2 mutant with ASB4 inhibits both differentiation and functional vascular responses.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, proteasome inhibitor treatment, degradation-resistant ID2 mutant rescue, endothelial co-culture functional assay, Asb4 knockout mouse placental phenotyping\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods including ubiquitination assay, proteasome-dependent degradation, dominant-negative rescue, in vivo KO phenotype, all in single study\",\n      \"pmids\": [\"24586788\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"ASB4 (Asb-4) interacts with GPS1 (CSN1) via its ankyrin repeat domain (independent of the SOCS box) and reduces GPS1 protein levels; co-expression of ASB4 with GPS1 inhibits c-Jun NH2-terminal kinase (JNK) activity and reduces insulin-stimulated IRS-1 serine 307 phosphorylation.\",\n      \"method\": \"Yeast two-hybrid screening, co-immunoprecipitation in vitro and in HEK293 cells, SOCS box deletion mutant, JNK activity assay, IRS-1 phosphorylation assay\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid plus reciprocal Co-IP plus deletion mutagenesis plus kinase activity assay, single lab\",\n      \"pmids\": [\"17276034\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"ASB4 co-localizes with IRS4 in hypothalamic POMC and NPY neurons, physically interacts with IRS4 (confirmed by Co-IP in cell lines and rat hypothalamic extracts), ubiquitinates IRS4 in a SOCS box-dependent manner, promotes IRS4 proteasomal degradation, and reduces both basal and insulin-stimulated AKT (Thr308) phosphorylation.\",\n      \"method\": \"In situ hybridization co-localization, co-immunoprecipitation (heterologous cells and endogenous hypothalamic extracts), SOCS box deletion mutant, ubiquitination assay, AKT phosphorylation assay\",\n      \"journal\": \"BMC neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods: co-IP in native tissue, ubiquitination assay, mutagenesis of SOCS box, downstream signaling readout, single lab but rigorous\",\n      \"pmids\": [\"21955513\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Overexpression of ASB4 specifically in POMC neurons of the arcuate nucleus increases food intake, reduces fat mass, increases lean mass, raises metabolic rate (O2 consumption and CO2 production), increases locomotor activity, and elevates POMC mRNA; ASB4 expression in the hypothalamus is regulated by insulin (paraventricular nucleus) and leptin (paraventricular nucleus and arcuate nucleus).\",\n      \"method\": \"Transgenic mouse model (POMC-Asb4), metabolic cage analysis, intracerebroventricular insulin injection, intraperitoneal leptin injection, quantitative gene expression\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean transgenic gain-of-function with defined metabolic phenotype, multiple physiological readouts, single lab\",\n      \"pmids\": [\"19934378\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ASB4 acts downstream of AgRP in the hypothalamus to regulate satiety and glucose homeostasis: hypothalamic Asb4 expression is suppressed by fasting in an AgRP-dependent manner; acute Asb4 knockdown causes hyperphagia via increased meal size; Asb4-deficient mice are resistant to calcitonin-induced meal termination and show reduced Calcr (calcitonin receptor) expression in neurons; POMC neuron-specific Asb4 deletion causes glucose intolerance independent of obesity.\",\n      \"method\": \"AgRP-knockout mice, acute siRNA knockdown, Asb4 global knockout, POMC-specific conditional knockout, calcitonin pharmacological challenge, qPCR for Calcr, metabolic and glucose tolerance testing\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple genetic models (global KO, POMC-specific KO, AgRP-KO epistasis), pharmacological challenge, defined molecular and behavioral phenotypes, single lab but multiple orthogonal approaches\",\n      \"pmids\": [\"35536884\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In the absence of ASB4, insulin (but not leptin) elevates ID2 protein levels post-transcriptionally in trophoblasts, implicating hyperinsulinemia in perturbing ASB4-mediated ID2 degradation and contributing to enhanced preeclampsia pathology.\",\n      \"method\": \"Asb4-null mice on high-fat diet, placental ID2 protein and mRNA quantification, insulin/leptin treatment of HTR8/SVneo human trophoblast cells\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo KO plus cell-based hormone treatment, protein vs. transcript comparison, single lab\",\n      \"pmids\": [\"36768469\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Asb4 is an imprinted gene showing differential expression between parthenogenetic and androgenetic mouse embryos, confirmed in normal diploid embryos from reciprocal F1 crosses.\",\n      \"method\": \"RIKEN cDNA microarray screen, reciprocal F1 cross validation by expression analysis\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — microarray with genetic cross validation, replicated in multiple cross directions but no mechanistic follow-up on imprinting mechanism\",\n      \"pmids\": [\"11820791\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ASB4 expression promotes migration and invasion of hepatocellular carcinoma (HCC) cells; suppression of ASB4 in HCC cell lines (PLC, MHCC97-L) reduces migration and invasion, while ectopic ASB4 expression in Hep3B cells enhances migration; ASB4 mRNA levels are negatively regulated by miR-200a through a validated binding site in the ASB4 3' UTR.\",\n      \"method\": \"siRNA knockdown and overexpression in HCC cell lines, migration/invasion assays, dual luciferase reporter assay for miR-200a 3' UTR binding\",\n      \"journal\": \"Bioscience trends\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — loss- and gain-of-function migration assays plus luciferase reporter for miRNA regulation, single lab\",\n      \"pmids\": [\"24815387\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ASB4 is a SOCS box-containing E3 ubiquitin ligase substrate-recognition subunit that assembles with elongin B/C/cullin/Roc to ubiquitinate and degrade specific substrates—including ID2 (in trophoblasts promoting placental vascular differentiation), IRS4 (in hypothalamic neurons modulating insulin signaling), and GPS1/CSN1 (suppressing JNK activity)—and whose activity is itself regulated by oxygen-dependent FIH-mediated hydroxylation; in the hypothalamus ASB4 acts downstream of AgRP to regulate satiety via calcitonin receptor expression and controls glucose homeostasis specifically in POMC neurons.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ASB4 is the substrate-recognition subunit of an elongin B/elongin C/cullin/Roc E3 ubiquitin ligase complex that targets specific proteins for ubiquitination and proteasomal degradation, coupling oxygen sensing and metabolic signaling to differentiation and energy homeostasis [#1]. Its ligase activity is regulated by oxygen tension: FIH hydroxylates an asparagine residue in ASB4 in normoxia, an oxygen-dependent modification linked to substrate binding and degradation [#0]. Through its SOCS box, ASB4 ubiquitinates and degrades the transcriptional regulator ID2 in trophoblasts, driving trophoblast differentiation and placental vascular patterning, and a degradation-resistant ID2 mutant blocks both responses [#2]; loss of ASB4 allows insulin to elevate ID2 post-transcriptionally, linking the pathway to preeclampsia pathology [#7]. In hypothalamic POMC and NPY neurons, ASB4 binds and ubiquitinates IRS4 in a SOCS box-dependent manner to dampen insulin/AKT signaling [#4], and acts downstream of AgRP to regulate satiety—being suppressed by fasting, required for calcitonin-induced meal termination via Calcr expression, and controlling glucose homeostasis in POMC neurons [#6]. Separately, ASB4 engages GPS1/CSN1 through its ankyrin-repeat domain, independent of the SOCS box, to suppress JNK activity and reduce IRS-1 serine phosphorylation [#3].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Established Asb4 as an imprinted gene, the first indication that its expression is under parent-of-origin genomic control.\",\n      \"evidence\": \"RIKEN cDNA microarray of parthenogenetic vs androgenetic embryos with reciprocal F1 cross validation\",\n      \"pmids\": [\"11820791\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No mechanism of imprinting (DMR, methylation) characterized\", \"No link drawn between imprinting and ASB4 protein function\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Defined the core molecular identity of ASB4 as a SOCS-box E3 ubiquitin ligase substrate-recognition subunit and tied its activity to oxygen via FIH hydroxylation, answering what biochemical machine ASB4 belongs to.\",\n      \"evidence\": \"SOCS box complex biochemistry, Co-IP and hydroxylation assay with FIH, ES cell overexpression with oxygen-dependent vascular differentiation readout\",\n      \"pmids\": [\"17636018\", \"11111040\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Endogenous substrate(s) in ES/vascular context not identified\", \"Functional consequence of hydroxylation on substrate degradation not directly demonstrated\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Revealed a SOCS-box-independent activity: ASB4 binds GPS1/CSN1 through its ankyrin repeats to suppress JNK and IRS-1 serine phosphorylation, expanding its role beyond canonical ligase function.\",\n      \"evidence\": \"Yeast two-hybrid, reciprocal Co-IP, SOCS box deletion mutant, JNK and IRS-1 phosphorylation assays in HEK293 cells\",\n      \"pmids\": [\"17276034\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether GPS1 reduction is ubiquitin-dependent not resolved\", \"In vivo relevance of the ASB4-GPS1-JNK axis untested\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Connected ASB4 to whole-animal energy balance by showing POMC-neuron overexpression alters food intake, body composition and metabolic rate, and that its hypothalamic expression responds to insulin and leptin.\",\n      \"evidence\": \"POMC-Asb4 transgenic mice, metabolic cage analysis, ICV insulin and IP leptin challenge, gene expression\",\n      \"pmids\": [\"19934378\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular substrate mediating the metabolic phenotype not identified in this study\", \"Gain-of-function only; loss-of-function not yet tested\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identified IRS4 as a neuronal substrate, providing a molecular substrate that links ASB4 ligase activity to attenuated insulin/AKT signaling in hypothalamic neurons.\",\n      \"evidence\": \"In situ co-localization, Co-IP in cells and endogenous hypothalamic extracts, SOCS box deletion mutant, ubiquitination and AKT phosphorylation assays\",\n      \"pmids\": [\"21955513\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo requirement of IRS4 degradation for the metabolic phenotype not established\", \"Relationship between IRS4 and downstream feeding circuits unresolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Established ID2 as a physiological substrate in trophoblasts and connected ASB4-mediated ID2 degradation to placental differentiation and vascular patterning, with in vivo KO validation.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, proteasome inhibition, degradation-resistant ID2 mutant rescue, endothelial co-culture, Asb4 knockout placental phenotyping\",\n      \"pmids\": [\"24586788\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether FIH hydroxylation gates ID2 degradation not tested here\", \"Upstream signals controlling ASB4 in trophoblasts not defined\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Implicated ASB4 in hepatocellular carcinoma cell motility and showed its mRNA is repressed by miR-200a, indicating a regulatory layer controlling ASB4 abundance.\",\n      \"evidence\": \"siRNA knockdown and overexpression in HCC cell lines, migration/invasion assays, dual luciferase reporter for miR-200a 3'UTR binding\",\n      \"pmids\": [\"24815387\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ligase substrate driving migration not identified\", \"No in vivo tumor model\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Placed ASB4 in a defined feeding circuit: it acts downstream of AgRP, is fasting-suppressed, mediates calcitonin-induced satiety via Calcr, and controls glucose homeostasis specifically in POMC neurons.\",\n      \"evidence\": \"AgRP-KO epistasis, acute siRNA knockdown, global and POMC-specific Asb4 KO, calcitonin challenge, qPCR for Calcr, glucose tolerance testing\",\n      \"pmids\": [\"35536884\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct ligase substrate controlling Calcr expression not identified\", \"Mechanism coupling ASB4 loss to glucose intolerance unresolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Linked hyperinsulinemia to disease by showing that in ASB4 absence insulin elevates ID2 post-transcriptionally in trophoblasts, connecting failed ID2 degradation to preeclampsia pathology.\",\n      \"evidence\": \"Asb4-null mice on high-fat diet, placental ID2 protein vs mRNA, insulin/leptin treatment of HTR8/SVneo trophoblasts\",\n      \"pmids\": [\"36768469\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal chain from insulin to ASB4 activity not mechanistically dissected\", \"Direct demonstration that ID2 stabilization drives the phenotype absent\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How oxygen-dependent FIH hydroxylation, substrate selection (ID2, IRS4, GPS1), and tissue context are integrated to direct ASB4 between vascular/placental differentiation and hypothalamic energy homeostasis remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of substrate recognition\", \"Hydroxylation-substrate degradation coupling not directly demonstrated for any substrate\", \"No unified in vivo test linking ligase activity to each phenotype\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 2, 4]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [1, 2, 4]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 2, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [3, 4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 2, 4]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 4, 6]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [1, 2]}\n    ],\n    \"complexes\": [\"elongin B/elongin C/cullin/Roc E3 ubiquitin ligase\"],\n    \"partners\": [\"FIH\", \"ID2\", \"IRS4\", \"GPS1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}