{"gene":"AAMP","run_date":"2026-06-09T22:02:35","timeline":{"discoveries":[{"year":1996,"finding":"AAMP is a 52 kDa protein containing immunoglobulin-type domains, WD40 repeats, a large acidic region with an acid box, a potential transmembrane region, serine/threonine phosphorylation sites, and a positively charged amino-terminal region with strong heparin binding potential (Kd = 14 pmol). Anti-AAMP antibody inhibits endothelial tube formation on Matrigel under cross-linking conditions, and AAMP is distributed both intracellularly and extracellularly in endothelial cell cultures.","method":"Sequence analysis, heparin binding assay, anti-AAMP antibody inhibition of endothelial tube formation, immunofluorescent staining","journal":"Laboratory investigation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional antibody inhibition assay plus localization data and binding constant, single lab with multiple orthogonal methods","pmids":["8683944"],"is_preprint":false},{"year":1996,"finding":"AAMP shares a common epitope (ESESES) with alpha-actinin and a fast skeletal muscle 23-kDa fiber protein; the epitope is continuous in AAMP but discontinuous/assembled in alpha-actinin. Thermolysin digestion destroys anti-P189 reactivity for alpha-actinin but not recombinant AAMP, demonstrating structural differences in how the epitope is presented.","method":"Peptide synthesis, polyclonal antibody generation, competition studies with peptide variants, thermolysin limited proteolysis, immunoperoxidase staining","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal biochemical methods (competition, proteolysis, staining) in a single lab","pmids":["8660919"],"is_preprint":false},{"year":1997,"finding":"An AAMP-derived peptide (P189, from the heparin-binding amino-terminal region) in aggregated particulate form binds heparin in a saturable manner (Kd = 306 pmol) and mediates heparin-sensitive cell binding/clustering; cell surface glycosaminoglycans are implicated. Tumor cell migration is partially inhibited by the peptide.","method":"Heparin binding assay, cell binding/clustering assay with inhibitors and heparin competition, peptide variant substitution studies, electron microscopy","journal":"Biotechnology and bioengineering","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — multiple functional assays and peptide variants in a single lab; mechanism linked to heparin/glycosaminoglycans","pmids":["18634104"],"is_preprint":false},{"year":2009,"finding":"AAMP was identified as a binding partner of Nod2 (NLR family) via yeast two-hybrid screen; co-immunoprecipitation from human cells confirmed the interaction and showed that an internal peptide of AAMP spanning three WD40 domains is sufficient for binding. AAMP is predominantly cytosolic in epithelial cells. Overexpression and siRNA knockdown demonstrated that AAMP modulates Nod2- and Nod1-mediated NF-κB activation in HEK293T cells.","method":"Yeast two-hybrid screen, co-immunoprecipitation, siRNA knockdown, overexpression, NF-κB reporter assay, immunofluorescence/subcellular localization","journal":"Molecular immunology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP confirming Y2H interaction, domain mapping, functional siRNA and OE assays with defined reporter readout in a single rigorous study","pmids":["19535145"],"is_preprint":false},{"year":2013,"finding":"Knockdown of AAMP (via hammerhead ribozyme transgene) in breast cancer cell lines reduced cell adhesion and cell growth (MCF-7) and suppressed cell invasion (MDA-MB-231), establishing a direct functional role for AAMP in breast cancer cell adhesion, growth, and invasion.","method":"Hammerhead ribozyme-mediated knockdown, in vitro cell adhesion, growth, and invasion assays","journal":"Anticancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean genetic knockdown with multiple defined cellular phenotype readouts, single lab, no pathway placement beyond loss-of-function","pmids":["23564791"],"is_preprint":false},{"year":2015,"finding":"AAMP localizes to cytoplasm and membrane in vascular endothelial cells, and is recruited by VEGF to cell membrane protrusions. siRNA knockdown and antibody blockade of AAMP impaired VEGF-induced endothelial tube formation and aortic ring angiogenic sprouting. AAMP knockdown reduced VEGF-induced actin stress fiber formation and collagen gel contraction. RhoA/Rho kinase signaling was identified as a downstream mediator of AAMP's role in endothelial cell migration and angiogenesis.","method":"siRNA knockdown, antibody blockade, tube formation assay, aortic ring assay, collagen gel contraction, immunofluorescence for localization/actin, RhoA/ROCK pathway analysis","journal":"Annals of biomedical engineering","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal functional assays (siRNA, antibody, in vitro and ex vivo angiogenesis), pathway placement via RhoA/ROCK, single lab","pmids":["26350504"],"is_preprint":false},{"year":2020,"finding":"AAMP interacts with CDC42 (confirmed by co-immunoprecipitation) and promotes CDC42 activation in NSCLC cells, resulting in formation of cellular protrusions. Mechanistically, AAMP enhances CDC42 activation by impairing the interaction between the GAP protein ARHGAP1 and CDC42, thereby preventing CDC42 inactivation.","method":"Co-immunoprecipitation, CDC42 activation assay, siRNA/overexpression, cell migration and invasion assays","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus functional pathway dissection (ARHGAP1 competition) in a single lab with multiple supporting assays","pmids":["33279622"],"is_preprint":false},{"year":2021,"finding":"AAMP binds directly to RhoA and suppresses its SMURF2-mediated ubiquitination and degradation, thereby stabilizing RhoA and increasing the level of active RhoA. SMURF2 was shown to act as an E3 ubiquitin ligase for RhoA. This AAMP-RhoA-SMURF2 axis promotes colorectal cancer cell migration and invasion.","method":"Co-immunoprecipitation (AAMP-RhoA binding), ubiquitination assay, siRNA knockdown, overexpression, cell migration and invasion assays","journal":"Molecular therapy oncolytics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, and functional epistasis with SMURF2, single lab with multiple orthogonal methods","pmids":["34901393"],"is_preprint":false},{"year":2022,"finding":"AAMP was identified as a binding partner of the co-stimulatory protein B7-H3 by yeast two-hybrid and mass spectrometry screens; binding was confirmed by bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation. On a functional level, AAMP modulates B7-H3-mediated effects on T-cell proliferation in a 3H-thymidine proliferation assay.","method":"Yeast two-hybrid, mass spectrometry, bimolecular fluorescence complementation (BiFC), co-immunoprecipitation, 3H-thymidine T-cell proliferation assay","journal":"Neuro-oncology advances","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — interaction confirmed by three orthogonal methods (Y2H, BiFC, Co-IP) plus functional assay, single lab","pmids":["35919070"],"is_preprint":false},{"year":2024,"finding":"Proteomics screen (following ubiquitination inhibition in primary human endothelial cells) identified AAMP as a negative regulator of endothelial barrier function whose turnover is controlled by ubiquitination. AAMP regulates the stability and activity of both RhoA and RhoB, and colocalizes with F-actin and cortactin at membrane ruffles, suggesting a role in F-actin dynamics.","method":"Proteomics (ubiquitination inhibitors MLN7243/MLN4924), endothelial barrier function assay, RhoA/RhoB activity and stability assays, co-localization with F-actin/cortactin by immunofluorescence","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proteomics-driven identification plus functional barrier assay and Rho GTPase mechanistic follow-up, single lab with multiple methods","pmids":["39404373"],"is_preprint":false}],"current_model":"AAMP is a cytosolic/membrane-associated WD40- and immunoglobulin-domain-containing protein that regulates cell migration, angiogenesis, and innate immunity: it binds and activates Rho GTPases (RhoA, RhoB, CDC42) by protecting RhoA from SMURF2-mediated ubiquitin-dependent degradation and by blocking ARHGAP1-mediated CDC42 inactivation, thereby promoting actin remodeling and cell motility; it also physically interacts with Nod2 (via its WD40 domains) to modulate Nod1/Nod2-driven NF-κB activation, and interacts with the co-stimulatory protein B7-H3 to influence T-cell responses."},"narrative":{"mechanistic_narrative":"AAMP is a WD40- and immunoglobulin-domain-containing protein, distributed both intracellularly and at the cell surface, that governs cell migration, angiogenesis, and innate immune signaling primarily by acting as a positive regulator of Rho-family GTPases and the actin cytoskeleton [PMID:8683944, PMID:26350504, PMID:39404373]. Its amino-terminal positively charged region binds heparin with high affinity and mediates heparin-sensitive, glycosaminoglycan-dependent cell binding and clustering, and anti-AAMP antibody blocks endothelial tube formation [PMID:8683944, PMID:18634104]. In vascular endothelial cells AAMP is recruited by VEGF to membrane protrusions and is required for VEGF-induced tube formation, aortic ring sprouting, actin stress fiber formation, and gel contraction through RhoA/Rho-kinase signaling [PMID:26350504]. Mechanistically, AAMP binds RhoA directly and protects it from SMURF2-mediated ubiquitination and degradation, thereby raising active RhoA levels [PMID:34901393], regulates the stability and activity of both RhoA and RhoB and colocalizes with F-actin and cortactin at membrane ruffles where it constrains endothelial barrier function [PMID:39404373], and binds CDC42 to promote its activation by impeding the ARHGAP1–CDC42 interaction [PMID:33279622]; collectively these activities drive cancer cell adhesion, growth, and invasion [PMID:23564791, PMID:33279622, PMID:34901393]. Independently of its cytoskeletal role, AAMP interacts via its WD40 domains with the NLR protein Nod2 and modulates Nod1/Nod2-driven NF-κB activation [PMID:19535145], and binds the co-stimulatory protein B7-H3 to influence T-cell proliferation [PMID:35919070].","teleology":[{"year":1996,"claim":"Established AAMP as a multidomain protein with both intracellular and extracellular distribution and a functional role in angiogenesis, defining the structural features that frame all later mechanistic work.","evidence":"Sequence analysis, heparin binding assay, anti-AAMP antibody inhibition of endothelial tube formation, and immunofluorescence in endothelial cells","pmids":["8683944"],"confidence":"Medium","gaps":["No direct demonstration of the molecular partners through which AAMP acts on tube formation","Functional role of the transmembrane region and acidic domain untested"]},{"year":1996,"claim":"Characterized a shared ESESES epitope between AAMP and alpha-actinin and showed it is presented differently, distinguishing AAMP structurally from a cytoskeletal protein it superficially resembles.","evidence":"Peptide competition, thermolysin limited proteolysis, and immunoperoxidase staining","pmids":["8660919"],"confidence":"Medium","gaps":["Epitope sharing does not establish a functional or interaction relationship with alpha-actinin"]},{"year":1997,"claim":"Mapped AAMP's heparin-binding and cell-clustering activity to its amino-terminal P189 peptide and linked it to cell-surface glycosaminoglycans, providing a basis for its extracellular adhesive function.","evidence":"Saturable heparin binding assay, GAG-dependent cell binding/clustering with inhibitors, peptide variant substitutions, and electron microscopy; tumor cell migration partially inhibited by peptide","pmids":["18634104"],"confidence":"Medium","gaps":["Activity demonstrated with aggregated synthetic peptide rather than full-length native protein","Physiological GAG receptor not identified"]},{"year":2009,"claim":"Connected AAMP to innate immunity by identifying it as a Nod2 interactor that tunes NF-κB activation, expanding AAMP's role beyond migration into NLR signaling.","evidence":"Yeast two-hybrid, reciprocal co-immunoprecipitation, WD40-domain mapping, siRNA/overexpression, and NF-κB reporter assays in HEK293T","pmids":["19535145"],"confidence":"High","gaps":["Position of AAMP within the Nod2 signaling complex not resolved","No in vivo confirmation of effect on inflammatory responses"]},{"year":2013,"claim":"Demonstrated that AAMP is functionally required for breast cancer cell adhesion, growth, and invasion, establishing a pro-tumorigenic loss-of-function phenotype.","evidence":"Hammerhead ribozyme knockdown with adhesion, growth, and invasion assays in MCF-7 and MDA-MB-231 cells","pmids":["23564791"],"confidence":"Medium","gaps":["No molecular pathway linked to the phenotypes in this study","No in vivo tumor model"]},{"year":2015,"claim":"Placed AAMP within VEGF-driven angiogenesis and identified RhoA/ROCK as the downstream effector pathway for its role in endothelial migration and actin remodeling.","evidence":"siRNA, antibody blockade, tube formation, aortic ring sprouting, collagen gel contraction, and actin/localization immunofluorescence in endothelial cells","pmids":["26350504"],"confidence":"Medium","gaps":["Direct biochemical link between AAMP and RhoA not yet established at this stage","Mechanism of VEGF-dependent recruitment to protrusions unknown"]},{"year":2020,"claim":"Defined a molecular mechanism by which AAMP activates CDC42 — by impairing the ARHGAP1–CDC42 interaction to block GAP-mediated inactivation — explaining protrusion formation in lung cancer cells.","evidence":"Co-immunoprecipitation, CDC42 activation assay, siRNA/overexpression, and migration/invasion assays in NSCLC cells","pmids":["33279622"],"confidence":"Medium","gaps":["Whether AAMP binds CDC42 and ARHGAP1 simultaneously or competitively not structurally resolved","Generality across cell types untested"]},{"year":2021,"claim":"Identified a second GTPase-stabilizing mechanism: AAMP binds RhoA directly and shields it from SMURF2-mediated ubiquitination, increasing active RhoA to drive colorectal cancer invasion.","evidence":"Co-immunoprecipitation, ubiquitination assay, SMURF2 epistasis, knockdown/overexpression, and migration/invasion assays","pmids":["34901393"],"confidence":"Medium","gaps":["Structural basis for how AAMP blocks SMURF2 access to RhoA unknown","Whether AAMP regulates SMURF2 activity broadly not addressed"]},{"year":2022,"claim":"Extended AAMP's interactome to the co-stimulatory protein B7-H3, implicating AAMP in modulation of T-cell responses.","evidence":"Yeast two-hybrid and mass spectrometry screens with BiFC and Co-IP validation, plus 3H-thymidine T-cell proliferation assay","pmids":["35919070"],"confidence":"Medium","gaps":["Mechanism by which AAMP affects B7-H3 signaling not defined","In vivo immune relevance untested"]},{"year":2024,"claim":"Unified AAMP's regulation as a ubiquitination-controlled negative regulator of endothelial barrier function acting on both RhoA and RhoB at actin-rich membrane ruffles.","evidence":"Ubiquitination-inhibitor proteomics (MLN7243/MLN4924), endothelial barrier assays, RhoA/RhoB activity and stability assays, and F-actin/cortactin colocalization in primary endothelial cells","pmids":["39404373"],"confidence":"Medium","gaps":["E3 ligase controlling AAMP turnover not identified","Mechanistic distinction between AAMP control of RhoA versus RhoB unresolved"]},{"year":null,"claim":"How AAMP's distinct activities — Rho/CDC42 GTPase regulation, extracellular heparin/GAG binding, Nod2/NF-κB modulation, and B7-H3-linked immune signaling — are integrated by a single protein, and whether these reflect separable domains or context-dependent functions, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model assigning functions to specific domains","No in vivo or knockout phenotype defining the dominant physiological role","Crosstalk between cytoskeletal and immune functions unexplored"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[6,7,9]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[5,9]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[3,8]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[0,2]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[3,5]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[5,9]},{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[0,2]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[5,9]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[5,6,7]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[3,8]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[4,6,7]}],"complexes":[],"partners":["RHOA","RHOB","CDC42","ARHGAP1","SMURF2","NOD2","CD276","CTTN"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q13685","full_name":"Angio-associated migratory cell protein","aliases":[],"length_aa":434,"mass_kda":46.8,"function":"Plays a role in angiogenesis and cell migration. In smooth muscle cell migration, may act through the RhoA pathway","subcellular_location":"Cell membrane; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q13685/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/AAMP","classification":"Common Essential","n_dependent_lines":1125,"n_total_lines":1208,"dependency_fraction":0.9312913907284768},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000127837","cell_line_id":"CID001050","localizations":[{"compartment":"cytoplasmic","grade":3},{"compartment":"nucleoplasm","grade":1}],"interactors":[{"gene":"SUB1","stoichiometry":10.0},{"gene":"ARGLU1","stoichiometry":4.0},{"gene":"DKC1","stoichiometry":4.0},{"gene":"SSRP1","stoichiometry":0.2},{"gene":"CWF19L2","stoichiometry":0.2},{"gene":"SUPT16H","stoichiometry":0.2},{"gene":"RPL10","stoichiometry":0.2},{"gene":"RSRC1","stoichiometry":0.2},{"gene":"SRSF6","stoichiometry":0.2},{"gene":"PRPF40A","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001050","total_profiled":1310},"omim":[{"mim_id":"603488","title":"ANGIO-ASSOCIATED MIGRATORY CELL PROTEIN; AAMP","url":"https://www.omim.org/entry/603488"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Microtubules","reliability":"Approved"},{"location":"Plasma membrane","reliability":"Additional"},{"location":"Cytokinetic bridge","reliability":"Additional"},{"location":"Primary cilium","reliability":"Additional"},{"location":"Primary cilium tip","reliability":"Additional"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/AAMP"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"Q13685","domains":[{"cath_id":"2.130.10.10","chopping":"83-212","consensus_level":"medium","plddt":97.1858,"start":83,"end":212}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13685","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q13685-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q13685-F1-predicted_aligned_error_v6.png","plddt_mean":84.56},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=AAMP","jax_strain_url":"https://www.jax.org/strain/search?query=AAMP"},"sequence":{"accession":"Q13685","fasta_url":"https://rest.uniprot.org/uniprotkb/Q13685.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q13685/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13685"}},"corpus_meta":[{"pmid":"19535145","id":"PMC_19535145","title":"A function for AAMP in Nod2-mediated NF-kappaB activation.","date":"2009","source":"Molecular immunology","url":"https://pubmed.ncbi.nlm.nih.gov/19535145","citation_count":30,"is_preprint":false},{"pmid":"26350504","id":"PMC_26350504","title":"AAMP Regulates Endothelial Cell Migration and Angiogenesis Through RhoA/Rho Kinase Signaling.","date":"2015","source":"Annals of biomedical engineering","url":"https://pubmed.ncbi.nlm.nih.gov/26350504","citation_count":22,"is_preprint":false},{"pmid":"8683944","id":"PMC_8683944","title":"AAMP, a conserved protein with immunoglobulin and WD40 domains, regulates endothelial tube formation in vitro.","date":"1996","source":"Laboratory investigation; a journal of technical methods and pathology","url":"https://pubmed.ncbi.nlm.nih.gov/8683944","citation_count":20,"is_preprint":false},{"pmid":"34901393","id":"PMC_34901393","title":"AAMP promotes colorectal cancermetastasis by suppressing SMURF2-mediatedubiquitination and degradation of RhoA.","date":"2021","source":"Molecular therapy oncolytics","url":"https://pubmed.ncbi.nlm.nih.gov/34901393","citation_count":19,"is_preprint":false},{"pmid":"27619413","id":"PMC_27619413","title":"The concept of allergen-associated molecular patterns (AAMP).","date":"2016","source":"Current opinion in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/27619413","citation_count":16,"is_preprint":false},{"pmid":"33279622","id":"PMC_33279622","title":"Angio-associated migratory cell protein (AAMP) interacts with cell division cycle 42 (CDC42) and enhances migration and invasion in human non-small cell lung cancer cells.","date":"2020","source":"Cancer letters","url":"https://pubmed.ncbi.nlm.nih.gov/33279622","citation_count":14,"is_preprint":false},{"pmid":"23564791","id":"PMC_23564791","title":"The impact of angio-associated migratory cell protein (AAMP) on breast cancer cells in vitro and its clinical significance.","date":"2013","source":"Anticancer research","url":"https://pubmed.ncbi.nlm.nih.gov/23564791","citation_count":13,"is_preprint":false},{"pmid":"35919070","id":"PMC_35919070","title":"AAMP is a binding partner of costimulatory human B7-H3.","date":"2022","source":"Neuro-oncology advances","url":"https://pubmed.ncbi.nlm.nih.gov/35919070","citation_count":13,"is_preprint":false},{"pmid":"8660919","id":"PMC_8660919","title":"AAMP, a newly identified protein, shares a common epitope with alpha-actinin and a fast skeletal muscle fiber protein.","date":"1996","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/8660919","citation_count":12,"is_preprint":false},{"pmid":"39404373","id":"PMC_39404373","title":"AAMP and MTSS1 Are Novel Negative Regulators of Endothelial Barrier Function Identified in a Proteomics Screen.","date":"2024","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/39404373","citation_count":3,"is_preprint":false},{"pmid":"37122729","id":"PMC_37122729","title":"A crucial exosome-related gene pair (AAMP and ABAT) is associated with inflammatory cells in intervertebral disc degeneration.","date":"2023","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/37122729","citation_count":2,"is_preprint":false},{"pmid":"18634104","id":"PMC_18634104","title":"The aggregated form of an AAMP derived peptide behaves as a heparin sensitive cell binding agent.","date":"1997","source":"Biotechnology and bioengineering","url":"https://pubmed.ncbi.nlm.nih.gov/18634104","citation_count":2,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7561,"output_tokens":2784,"usd":0.032222,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10081,"output_tokens":3746,"usd":0.072027,"stage2_stop_reason":"end_turn"},"total_usd":0.104249,"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\": 1996,\n      \"finding\": \"AAMP is a 52 kDa protein containing immunoglobulin-type domains, WD40 repeats, a large acidic region with an acid box, a potential transmembrane region, serine/threonine phosphorylation sites, and a positively charged amino-terminal region with strong heparin binding potential (Kd = 14 pmol). Anti-AAMP antibody inhibits endothelial tube formation on Matrigel under cross-linking conditions, and AAMP is distributed both intracellularly and extracellularly in endothelial cell cultures.\",\n      \"method\": \"Sequence analysis, heparin binding assay, anti-AAMP antibody inhibition of endothelial tube formation, immunofluorescent staining\",\n      \"journal\": \"Laboratory investigation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional antibody inhibition assay plus localization data and binding constant, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"8683944\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"AAMP shares a common epitope (ESESES) with alpha-actinin and a fast skeletal muscle 23-kDa fiber protein; the epitope is continuous in AAMP but discontinuous/assembled in alpha-actinin. Thermolysin digestion destroys anti-P189 reactivity for alpha-actinin but not recombinant AAMP, demonstrating structural differences in how the epitope is presented.\",\n      \"method\": \"Peptide synthesis, polyclonal antibody generation, competition studies with peptide variants, thermolysin limited proteolysis, immunoperoxidase staining\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal biochemical methods (competition, proteolysis, staining) in a single lab\",\n      \"pmids\": [\"8660919\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"An AAMP-derived peptide (P189, from the heparin-binding amino-terminal region) in aggregated particulate form binds heparin in a saturable manner (Kd = 306 pmol) and mediates heparin-sensitive cell binding/clustering; cell surface glycosaminoglycans are implicated. Tumor cell migration is partially inhibited by the peptide.\",\n      \"method\": \"Heparin binding assay, cell binding/clustering assay with inhibitors and heparin competition, peptide variant substitution studies, electron microscopy\",\n      \"journal\": \"Biotechnology and bioengineering\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — multiple functional assays and peptide variants in a single lab; mechanism linked to heparin/glycosaminoglycans\",\n      \"pmids\": [\"18634104\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"AAMP was identified as a binding partner of Nod2 (NLR family) via yeast two-hybrid screen; co-immunoprecipitation from human cells confirmed the interaction and showed that an internal peptide of AAMP spanning three WD40 domains is sufficient for binding. AAMP is predominantly cytosolic in epithelial cells. Overexpression and siRNA knockdown demonstrated that AAMP modulates Nod2- and Nod1-mediated NF-κB activation in HEK293T cells.\",\n      \"method\": \"Yeast two-hybrid screen, co-immunoprecipitation, siRNA knockdown, overexpression, NF-κB reporter assay, immunofluorescence/subcellular localization\",\n      \"journal\": \"Molecular immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP confirming Y2H interaction, domain mapping, functional siRNA and OE assays with defined reporter readout in a single rigorous study\",\n      \"pmids\": [\"19535145\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Knockdown of AAMP (via hammerhead ribozyme transgene) in breast cancer cell lines reduced cell adhesion and cell growth (MCF-7) and suppressed cell invasion (MDA-MB-231), establishing a direct functional role for AAMP in breast cancer cell adhesion, growth, and invasion.\",\n      \"method\": \"Hammerhead ribozyme-mediated knockdown, in vitro cell adhesion, growth, and invasion assays\",\n      \"journal\": \"Anticancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean genetic knockdown with multiple defined cellular phenotype readouts, single lab, no pathway placement beyond loss-of-function\",\n      \"pmids\": [\"23564791\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"AAMP localizes to cytoplasm and membrane in vascular endothelial cells, and is recruited by VEGF to cell membrane protrusions. siRNA knockdown and antibody blockade of AAMP impaired VEGF-induced endothelial tube formation and aortic ring angiogenic sprouting. AAMP knockdown reduced VEGF-induced actin stress fiber formation and collagen gel contraction. RhoA/Rho kinase signaling was identified as a downstream mediator of AAMP's role in endothelial cell migration and angiogenesis.\",\n      \"method\": \"siRNA knockdown, antibody blockade, tube formation assay, aortic ring assay, collagen gel contraction, immunofluorescence for localization/actin, RhoA/ROCK pathway analysis\",\n      \"journal\": \"Annals of biomedical engineering\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal functional assays (siRNA, antibody, in vitro and ex vivo angiogenesis), pathway placement via RhoA/ROCK, single lab\",\n      \"pmids\": [\"26350504\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"AAMP interacts with CDC42 (confirmed by co-immunoprecipitation) and promotes CDC42 activation in NSCLC cells, resulting in formation of cellular protrusions. Mechanistically, AAMP enhances CDC42 activation by impairing the interaction between the GAP protein ARHGAP1 and CDC42, thereby preventing CDC42 inactivation.\",\n      \"method\": \"Co-immunoprecipitation, CDC42 activation assay, siRNA/overexpression, cell migration and invasion assays\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus functional pathway dissection (ARHGAP1 competition) in a single lab with multiple supporting assays\",\n      \"pmids\": [\"33279622\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"AAMP binds directly to RhoA and suppresses its SMURF2-mediated ubiquitination and degradation, thereby stabilizing RhoA and increasing the level of active RhoA. SMURF2 was shown to act as an E3 ubiquitin ligase for RhoA. This AAMP-RhoA-SMURF2 axis promotes colorectal cancer cell migration and invasion.\",\n      \"method\": \"Co-immunoprecipitation (AAMP-RhoA binding), ubiquitination assay, siRNA knockdown, overexpression, cell migration and invasion assays\",\n      \"journal\": \"Molecular therapy oncolytics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, and functional epistasis with SMURF2, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"34901393\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"AAMP was identified as a binding partner of the co-stimulatory protein B7-H3 by yeast two-hybrid and mass spectrometry screens; binding was confirmed by bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation. On a functional level, AAMP modulates B7-H3-mediated effects on T-cell proliferation in a 3H-thymidine proliferation assay.\",\n      \"method\": \"Yeast two-hybrid, mass spectrometry, bimolecular fluorescence complementation (BiFC), co-immunoprecipitation, 3H-thymidine T-cell proliferation assay\",\n      \"journal\": \"Neuro-oncology advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — interaction confirmed by three orthogonal methods (Y2H, BiFC, Co-IP) plus functional assay, single lab\",\n      \"pmids\": [\"35919070\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Proteomics screen (following ubiquitination inhibition in primary human endothelial cells) identified AAMP as a negative regulator of endothelial barrier function whose turnover is controlled by ubiquitination. AAMP regulates the stability and activity of both RhoA and RhoB, and colocalizes with F-actin and cortactin at membrane ruffles, suggesting a role in F-actin dynamics.\",\n      \"method\": \"Proteomics (ubiquitination inhibitors MLN7243/MLN4924), endothelial barrier function assay, RhoA/RhoB activity and stability assays, co-localization with F-actin/cortactin by immunofluorescence\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proteomics-driven identification plus functional barrier assay and Rho GTPase mechanistic follow-up, single lab with multiple methods\",\n      \"pmids\": [\"39404373\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"AAMP is a cytosolic/membrane-associated WD40- and immunoglobulin-domain-containing protein that regulates cell migration, angiogenesis, and innate immunity: it binds and activates Rho GTPases (RhoA, RhoB, CDC42) by protecting RhoA from SMURF2-mediated ubiquitin-dependent degradation and by blocking ARHGAP1-mediated CDC42 inactivation, thereby promoting actin remodeling and cell motility; it also physically interacts with Nod2 (via its WD40 domains) to modulate Nod1/Nod2-driven NF-κB activation, and interacts with the co-stimulatory protein B7-H3 to influence T-cell responses.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"AAMP is a WD40- and immunoglobulin-domain-containing protein, distributed both intracellularly and at the cell surface, that governs cell migration, angiogenesis, and innate immune signaling primarily by acting as a positive regulator of Rho-family GTPases and the actin cytoskeleton [#0, #5, #9]. Its amino-terminal positively charged region binds heparin with high affinity and mediates heparin-sensitive, glycosaminoglycan-dependent cell binding and clustering, and anti-AAMP antibody blocks endothelial tube formation [#0, #2]. In vascular endothelial cells AAMP is recruited by VEGF to membrane protrusions and is required for VEGF-induced tube formation, aortic ring sprouting, actin stress fiber formation, and gel contraction through RhoA/Rho-kinase signaling [#5]. Mechanistically, AAMP binds RhoA directly and protects it from SMURF2-mediated ubiquitination and degradation, thereby raising active RhoA levels [#7], regulates the stability and activity of both RhoA and RhoB and colocalizes with F-actin and cortactin at membrane ruffles where it constrains endothelial barrier function [#9], and binds CDC42 to promote its activation by impeding the ARHGAP1–CDC42 interaction [#6]; collectively these activities drive cancer cell adhesion, growth, and invasion [#4, #6, #7]. Independently of its cytoskeletal role, AAMP interacts via its WD40 domains with the NLR protein Nod2 and modulates Nod1/Nod2-driven NF-\\u03baB activation [#3], and binds the co-stimulatory protein B7-H3 to influence T-cell proliferation [#8].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Established AAMP as a multidomain protein with both intracellular and extracellular distribution and a functional role in angiogenesis, defining the structural features that frame all later mechanistic work.\",\n      \"evidence\": \"Sequence analysis, heparin binding assay, anti-AAMP antibody inhibition of endothelial tube formation, and immunofluorescence in endothelial cells\",\n      \"pmids\": [\"8683944\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct demonstration of the molecular partners through which AAMP acts on tube formation\", \"Functional role of the transmembrane region and acidic domain untested\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Characterized a shared ESESES epitope between AAMP and alpha-actinin and showed it is presented differently, distinguishing AAMP structurally from a cytoskeletal protein it superficially resembles.\",\n      \"evidence\": \"Peptide competition, thermolysin limited proteolysis, and immunoperoxidase staining\",\n      \"pmids\": [\"8660919\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Epitope sharing does not establish a functional or interaction relationship with alpha-actinin\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Mapped AAMP's heparin-binding and cell-clustering activity to its amino-terminal P189 peptide and linked it to cell-surface glycosaminoglycans, providing a basis for its extracellular adhesive function.\",\n      \"evidence\": \"Saturable heparin binding assay, GAG-dependent cell binding/clustering with inhibitors, peptide variant substitutions, and electron microscopy; tumor cell migration partially inhibited by peptide\",\n      \"pmids\": [\"18634104\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Activity demonstrated with aggregated synthetic peptide rather than full-length native protein\", \"Physiological GAG receptor not identified\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Connected AAMP to innate immunity by identifying it as a Nod2 interactor that tunes NF-\\u03baB activation, expanding AAMP's role beyond migration into NLR signaling.\",\n      \"evidence\": \"Yeast two-hybrid, reciprocal co-immunoprecipitation, WD40-domain mapping, siRNA/overexpression, and NF-\\u03baB reporter assays in HEK293T\",\n      \"pmids\": [\"19535145\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Position of AAMP within the Nod2 signaling complex not resolved\", \"No in vivo confirmation of effect on inflammatory responses\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Demonstrated that AAMP is functionally required for breast cancer cell adhesion, growth, and invasion, establishing a pro-tumorigenic loss-of-function phenotype.\",\n      \"evidence\": \"Hammerhead ribozyme knockdown with adhesion, growth, and invasion assays in MCF-7 and MDA-MB-231 cells\",\n      \"pmids\": [\"23564791\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No molecular pathway linked to the phenotypes in this study\", \"No in vivo tumor model\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Placed AAMP within VEGF-driven angiogenesis and identified RhoA/ROCK as the downstream effector pathway for its role in endothelial migration and actin remodeling.\",\n      \"evidence\": \"siRNA, antibody blockade, tube formation, aortic ring sprouting, collagen gel contraction, and actin/localization immunofluorescence in endothelial cells\",\n      \"pmids\": [\"26350504\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct biochemical link between AAMP and RhoA not yet established at this stage\", \"Mechanism of VEGF-dependent recruitment to protrusions unknown\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Defined a molecular mechanism by which AAMP activates CDC42 — by impairing the ARHGAP1–CDC42 interaction to block GAP-mediated inactivation — explaining protrusion formation in lung cancer cells.\",\n      \"evidence\": \"Co-immunoprecipitation, CDC42 activation assay, siRNA/overexpression, and migration/invasion assays in NSCLC cells\",\n      \"pmids\": [\"33279622\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether AAMP binds CDC42 and ARHGAP1 simultaneously or competitively not structurally resolved\", \"Generality across cell types untested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identified a second GTPase-stabilizing mechanism: AAMP binds RhoA directly and shields it from SMURF2-mediated ubiquitination, increasing active RhoA to drive colorectal cancer invasion.\",\n      \"evidence\": \"Co-immunoprecipitation, ubiquitination assay, SMURF2 epistasis, knockdown/overexpression, and migration/invasion assays\",\n      \"pmids\": [\"34901393\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis for how AAMP blocks SMURF2 access to RhoA unknown\", \"Whether AAMP regulates SMURF2 activity broadly not addressed\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Extended AAMP's interactome to the co-stimulatory protein B7-H3, implicating AAMP in modulation of T-cell responses.\",\n      \"evidence\": \"Yeast two-hybrid and mass spectrometry screens with BiFC and Co-IP validation, plus 3H-thymidine T-cell proliferation assay\",\n      \"pmids\": [\"35919070\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which AAMP affects B7-H3 signaling not defined\", \"In vivo immune relevance untested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Unified AAMP's regulation as a ubiquitination-controlled negative regulator of endothelial barrier function acting on both RhoA and RhoB at actin-rich membrane ruffles.\",\n      \"evidence\": \"Ubiquitination-inhibitor proteomics (MLN7243/MLN4924), endothelial barrier assays, RhoA/RhoB activity and stability assays, and F-actin/cortactin colocalization in primary endothelial cells\",\n      \"pmids\": [\"39404373\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"E3 ligase controlling AAMP turnover not identified\", \"Mechanistic distinction between AAMP control of RhoA versus RhoB unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How AAMP's distinct activities — Rho/CDC42 GTPase regulation, extracellular heparin/GAG binding, Nod2/NF-\\u03baB modulation, and B7-H3-linked immune signaling — are integrated by a single protein, and whether these reflect separable domains or context-dependent functions, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model assigning functions to specific domains\", \"No in vivo or knockout phenotype defining the dominant physiological role\", \"Crosstalk between cytoskeletal and immune functions unexplored\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [6, 7, 9]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [5, 9]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [3, 8]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [0, 2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [3, 5]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [5, 9]},\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [0, 2]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [5, 9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [5, 6, 7]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [3, 8]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [4, 6, 7]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"RHOA\", \"RHOB\", \"CDC42\", \"ARHGAP1\", \"SMURF2\", \"NOD2\", \"CD276\", \"CTTN\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}