{"gene":"USP6NL","run_date":"2026-06-11T09:02:06","timeline":{"discoveries":[{"year":1996,"finding":"RN-tre (USP6NL) was identified as an 828 amino acid protein that binds specifically to the SH3 domain of Eps8 with high affinity (Kd 10^-8 to 10^-7 M), both in vitro and in vivo; the TrH (Tre Homology) domain in its N-terminus has protein-binding properties in vitro. A C-terminal truncated mutant conferred proliferative advantage and reduced serum requirement to NIH3T3 fibroblasts.","method":"In vitro binding assays, co-immunoprecipitation, NIH3T3 transformation assay","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal in vitro and in vivo binding with quantified Kd, functional truncation mutant, single lab","pmids":["8700527"],"is_preprint":false},{"year":1996,"finding":"RN-tre (USP6NL) maps to chromosomal region 10p13, is ubiquitously expressed, and the tre oncogene was shown to be a fusion product of a 5' element homologous to RN-tre and a 3' element encoding a deubiquitinating enzyme; the TrH domain is conserved from yeast to mammals and has protein-binding properties in vitro.","method":"cDNA cloning, chromosomal mapping, in vitro protein binding assay","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct experimental mapping and domain characterization, single lab, two methods","pmids":["8700515"],"is_preprint":false},{"year":2002,"finding":"RN-tre (USP6NL) associates with the Grb2 adaptor protein via Grb2's SH3 domains binding to proline-rich sequences in RN-tre, both in vitro and in vivo; this interaction is constitutive and independent of Eps8. EGF stimulates Grb2-dependent recruitment of RN-tre to the EGFR. Overexpression of RN-tre blocks internalization of EGFR, and a Grb2 mutant deficient in RN-tre binding is not blocked by RN-tre overexpression, demonstrating that RN-tre inhibits EGFR endocytosis through Grb2-mediated receptor binding.","method":"Co-immunoprecipitation (in vitro and in vivo), overexpression/dominant-negative assays, fluorescence microscopy","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP in vitro and in vivo, mechanistic dissection with Grb2 binding-deficient mutant, replicated across multiple experiments","pmids":["12399475"],"is_preprint":false},{"year":2007,"finding":"RN-tre (USP6NL) is a physiological substrate of the human Cdc14A dual-specificity phosphatase. RN-tre undergoes cell cycle-dependent phosphorylation peaking at mitosis, phosphorylated by cyclin-dependent kinase; hCdc14A dephosphorylates RN-tre both in vitro and in vivo. RN-tre phosphorylation is required for efficient hCdc14A binding and finely modulates RN-tre's GAP catalytic activity.","method":"Genetic substrate-trapping (catalytically inactive hCdc14A C278S mutant), Co-immunoprecipitation, in vitro phosphatase assay, cell cycle synchronization, CDK phosphorylation assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — substrate-trapping combined with in vitro phosphatase assay and in vivo validation, multiple orthogonal methods in a single rigorous study","pmids":["17371873"],"is_preprint":false},{"year":2013,"finding":"RN-tre (USP6NL) localizes to focal adhesions and Rab5-positive vesicles associated with focal adhesions undergoing rapid remodeling. RN-tre acts as a GAP for Rab5 and Rab43 and inhibits endocytosis of β1 (but not β3) integrins, delaying focal adhesion turnover and impairing β1-dependent chemotactic cell migration. All effects require GAP activity and are Rab5-dependent.","method":"Live-cell imaging, loss-of-function (siRNA), rescue with GAP-dead mutant, integrin endocytosis assay, chemotaxis assay, fluorescence microscopy","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (live imaging, loss-of-function, GAP-dead mutant rescue, functional migration assay), integrin selectivity established","pmids":["24239119"],"is_preprint":false},{"year":2018,"finding":"High USP6NL (RN-tre) levels in breast cancer cells delay endocytosis and degradation of EGFR, causing chronic AKT activation. In turn, activated AKT stabilizes GLUT1 at the plasma membrane, increasing aerobic glycolysis. Depletion of USP6NL accelerated EGFR/AKT downregulation and GLUT1 degradation, impairing proliferation specifically in USP6NL-high cancer cells.","method":"siRNA knockdown, EGFR endocytosis assay, western blot (phospho-AKT), GLUT1 membrane fractionation, glucose uptake assay, cell proliferation assay","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods establishing the USP6NL→EGFR→AKT→GLUT1 axis with knockdown and functional metabolic readouts","pmids":["29691252"],"is_preprint":false},{"year":2020,"finding":"In Drosophila S2 cells, depletion of RN-tre (ortholog of USP6NL) leads to a punctate non-muscle myosin II (NMII) RLC phenotype, decreased active Rho1, and decreased phosphomyosin; constitutively active Rho or Rho-kinase (Rok) rescues this phenotype. RN-tre regulation of NMII is only partially dependent on GAP activity (a GAP-dead RN-tre partially restores phosphomyosin, and constitutively active Rab substrates do not recapitulate the NMII phenotype), suggesting RN-tre links the secretion/endocytic machinery to actomyosin contractility via Rho1 signaling.","method":"RNAi screen in Drosophila S2 cells, immunostaining (phosphomyosin), GAP-dead mutant, constitutively active Rho/Rok rescue, actin retrograde flow assay, contractility assay","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple methods (RNAi, mutant rescue, functional assays) in Drosophila ortholog, single lab","pmids":["32816624"],"is_preprint":false},{"year":2025,"finding":"The crystal structure of the RN-Tre (USP6NL)–Rab43 complex reveals a bipartite recognition mechanism: the N-terminal TBC subdomain catalytically remodels Rab43 Switch regions (via RQ-dual finger mechanism), while the C-terminal subdomain engages Switch II and reorients the hydrophobic triad to confer substrate specificity. Leu146 and C-terminal residues are key specificity determinants, and the same structural analysis identified Rab19 as an additional substrate. Disease-associated RN-Tre mutations impair GAP activity, resulting in aberrant Golgi architecture and endocytic trafficking.","method":"X-ray crystallography (crystal structure of RN-Tre–Rab43 complex), mutational analysis, in vitro GAP activity assay, functional assays for Golgi morphology and endocytic trafficking","journal":"International journal of biological macromolecules","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with mutagenesis and in vitro functional validation in a single rigorous study","pmids":["41401861"],"is_preprint":false}],"current_model":"USP6NL (RN-tre) is a TBC-domain Rab-GTPase-activating protein (GAP) that inactivates Rab5 and Rab43 (and Rab19) via a bipartite RQ-dual-finger mechanism structurally defined at atomic resolution; it is recruited to activated EGFR via constitutive association with Grb2, where it negatively regulates Rab5-dependent endocytosis, thereby prolonging EGFR/AKT signaling and stabilizing GLUT1 at the plasma membrane to fuel glycolysis; at focal adhesions it selectively inhibits β1-integrin endocytosis in a Rab5-GAP-dependent manner to control focal adhesion turnover and chemotactic migration; its GAP activity and cellular localization are regulated by CDK-dependent mitotic phosphorylation that is reversed by the hCdc14A phosphatase; and it also cross-talks with the Rho1–ROCK–myosin II contractility axis partly independently of its GAP activity."},"narrative":{"mechanistic_narrative":"USP6NL (RN-tre) is a TBC-domain Rab-GTPase-activating protein that controls membrane trafficking by inactivating specific Rab GTPases, and through this activity it tunes receptor signaling, focal adhesion dynamics, and Golgi architecture [PMID:12399475, PMID:24239119, PMID:41401861]. Structurally, it engages its substrate Rab43 through a bipartite recognition mechanism in which an N-terminal TBC subdomain catalytically remodels the Rab Switch regions via an RQ-dual-finger arrangement while a C-terminal subdomain enforces substrate specificity; this analysis defined Rab19 as an additional substrate and showed that disease-associated mutations impair GAP activity to produce aberrant Golgi architecture and endocytic trafficking [PMID:41401861]. USP6NL acts as a GAP for Rab5 and Rab43 [PMID:24239119]. It is recruited to activated EGFR through a constitutive, Eps8-independent association with the Grb2 adaptor, where it inhibits Rab5-dependent EGFR endocytosis; in breast cancer cells with high USP6NL this delays EGFR/AKT downregulation, stabilizes GLUT1 at the plasma membrane, and sustains aerobic glycolysis and proliferation [PMID:12399475, PMID:29691252]. At focal adhesions and associated Rab5-positive vesicles, USP6NL selectively blocks endocytosis of β1 (but not β3) integrins in a GAP- and Rab5-dependent manner, delaying focal adhesion turnover and impairing chemotactic migration [PMID:24239119]. Its GAP activity is cell-cycle regulated through CDK-dependent mitotic phosphorylation that is reversed by the hCdc14A dual-specificity phosphatase [PMID:17371873], and in Drosophila it additionally links the trafficking machinery to Rho1–ROCK–myosin II contractility partly independently of its GAP activity [PMID:32816624].","teleology":[{"year":1996,"claim":"Establishing USP6NL's molecular identity and first protein partner addressed what this newly cloned protein is and how it might act, revealing an Eps8-binding protein with transforming potential and a conserved TrH domain.","evidence":"cDNA cloning, chromosomal mapping, in vitro/in vivo SH3-binding assays, and NIH3T3 transformation assay","pmids":["8700527","8700515"],"confidence":"Medium","gaps":["No enzymatic activity or substrate defined at this stage","Mechanism of transformation by the truncation mutant unresolved","Cellular pathway context unknown"]},{"year":2002,"claim":"Identifying a constitutive Grb2 association resolved how USP6NL is targeted to its site of action, showing EGF-driven Grb2-dependent recruitment to EGFR that blocks receptor internalization.","evidence":"Reciprocal Co-IP in vitro and in vivo, Grb2 binding-deficient mutant, overexpression/dominant-negative assays, fluorescence microscopy","pmids":["12399475"],"confidence":"High","gaps":["Did not define the Rab substrate underlying the endocytic block","Physiological versus overexpression-dependent effects not separated"]},{"year":2007,"claim":"Demonstrating cell-cycle phosphorylation and hCdc14A-mediated dephosphorylation answered how USP6NL's catalytic activity is temporally regulated, linking its GAP function to mitotic control.","evidence":"Substrate-trapping with catalytically inactive hCdc14A, in vitro phosphatase assay, CDK phosphorylation assay, cell cycle synchronization, Co-IP","pmids":["17371873"],"confidence":"High","gaps":["Specific phosphosites and their individual functional consequences not fully mapped","Trafficking consequence of mitotic regulation not directly tested"]},{"year":2013,"claim":"Defining USP6NL as a Rab5/Rab43 GAP at focal adhesions established its substrate specificity and a concrete cellular role, showing selective inhibition of β1-integrin endocytosis controls focal adhesion turnover and migration.","evidence":"Live-cell imaging, siRNA loss-of-function, GAP-dead rescue, integrin endocytosis and chemotaxis assays","pmids":["24239119"],"confidence":"High","gaps":["Basis for β1 versus β3 integrin selectivity not structurally explained","Whether Grb2 recruitment operates at focal adhesions not addressed"]},{"year":2018,"claim":"Connecting USP6NL to a EGFR→AKT→GLUT1 metabolic axis explained how its endocytic control feeds tumor metabolism, identifying it as a driver of aerobic glycolysis and proliferation in USP6NL-high breast cancer.","evidence":"siRNA knockdown, EGFR endocytosis assay, phospho-AKT western blot, GLUT1 membrane fractionation, glucose uptake and proliferation assays","pmids":["29691252"],"confidence":"High","gaps":["Direct GAP-dependence of the metabolic phenotype not isolated with a GAP-dead mutant","In vivo tumor relevance not established here"]},{"year":2020,"claim":"An RNAi screen in Drosophila revealed a GAP-partially-independent link between USP6NL and Rho1–ROCK–myosin II contractility, expanding its role beyond canonical Rab inactivation.","evidence":"RNAi in S2 cells, phosphomyosin immunostaining, GAP-dead mutant, constitutively active Rho/Rok rescue, contractility and actin flow assays","pmids":["32816624"],"confidence":"Medium","gaps":["GAP-independent mechanism on Rho1 not molecularly defined","Conservation of this function in mammalian cells not confirmed","Single lab, ortholog-based"]},{"year":2025,"claim":"The RN-Tre–Rab43 crystal structure resolved the long-open question of how USP6NL achieves catalysis and substrate selectivity, defining a bipartite RQ-dual-finger mechanism and linking disease mutations to impaired GAP activity and Golgi/endocytic defects.","evidence":"X-ray crystallography of the RN-Tre–Rab43 complex, mutational analysis, in vitro GAP assays, Golgi morphology and trafficking assays","pmids":["41401861"],"confidence":"High","gaps":["Structural basis for Rab5 recognition not directly solved","Specific disease and inheritance pattern not detailed in this analysis"]},{"year":null,"claim":"How USP6NL's distinct functional contexts — EGFR/Grb2 signaling, focal-adhesion integrin trafficking, mitotic phosphoregulation, Golgi maintenance, and Rho1-contractility crosstalk — are coordinated by a single GAP within one cell remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model integrating its multiple substrates and localizations","Mechanism switching GAP-dependent versus GAP-independent functions unknown","In vivo physiological hierarchy of these roles undetermined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[4,7]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[2]}],"localization":[{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[4]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[7]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[2,5]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[2,4]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,5]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[5]}],"complexes":[],"partners":["GRB2","EPS8","CDC14A","RAB5A","RAB43","EGFR"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q92738","full_name":"USP6 N-terminal-like protein","aliases":["Related to the N-terminus of tre","RN-tre"],"length_aa":828,"mass_kda":94.1,"function":"Acts as a GTPase-activating protein for RAB43, RAB5A and RAB30 (PubMed:11099046, PubMed:16086013, PubMed:17562788, PubMed:17684057). Involved in receptor trafficking. In complex with EPS8 inhibits internalization of EGFR. Involved in retrograde transport from the endocytic pathway to the Golgi apparatus. Involved in the transport of Shiga toxin from early and recycling endosomes to the trans-Golgi network. Required for structural integrity of the Golgi complex","subcellular_location":"Golgi apparatus; Cytoplasmic vesicle","url":"https://www.uniprot.org/uniprotkb/Q92738/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/USP6NL","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/USP6NL","total_profiled":1310},"omim":[{"mim_id":"605405","title":"USP6 N-TERMINAL-LIKE; USP6NL","url":"https://www.omim.org/entry/605405"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/USP6NL"},"hgnc":{"alias_symbol":["RNTRE","KIAA0019","TRE2NL","RN-tre"],"prev_symbol":["USP6NL-IT1"]},"alphafold":{"accession":"Q92738","domains":[{"cath_id":"1.10.8.270","chopping":"2-214","consensus_level":"high","plddt":91.4377,"start":2,"end":214},{"cath_id":"1.10.472.80","chopping":"230-353","consensus_level":"high","plddt":96.7457,"start":230,"end":353}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q92738","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q92738-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q92738-F1-predicted_aligned_error_v6.png","plddt_mean":61.94},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=USP6NL","jax_strain_url":"https://www.jax.org/strain/search?query=USP6NL"},"sequence":{"accession":"Q92738","fasta_url":"https://rest.uniprot.org/uniprotkb/Q92738.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q92738/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q92738"}},"corpus_meta":[{"pmid":"29691252","id":"PMC_29691252","title":"High USP6NL Levels in Breast Cancer Sustain Chronic AKT Phosphorylation and GLUT1 Stability Fueling Aerobic Glycolysis.","date":"2018","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/29691252","citation_count":62,"is_preprint":false},{"pmid":"12399475","id":"PMC_12399475","title":"Endocytosis of epidermal growth factor receptor regulated by Grb2-mediated recruitment of the Rab5 GTPase-activating protein RN-tre.","date":"2002","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12399475","citation_count":53,"is_preprint":false},{"pmid":"24239119","id":"PMC_24239119","title":"The GTPase-activating protein RN-tre controls focal adhesion turnover and cell migration.","date":"2013","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/24239119","citation_count":46,"is_preprint":false},{"pmid":"8700527","id":"PMC_8700527","title":"RN-tre specifically binds to the SH3 domain of eps8 with high affinity and confers growth advantage to NIH3T3 upon carboxy-terminal truncation.","date":"1996","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/8700527","citation_count":33,"is_preprint":false},{"pmid":"31015802","id":"PMC_31015802","title":"Tre2 (USP6NL) promotes colorectal cancer cell proliferation via Wnt/β-catenin pathway.","date":"2019","source":"Cancer cell international","url":"https://pubmed.ncbi.nlm.nih.gov/31015802","citation_count":27,"is_preprint":false},{"pmid":"17371873","id":"PMC_17371873","title":"Regulation of the Rab5 GTPase-activating protein RN-tre by the dual specificity phosphatase Cdc14A in human cells.","date":"2007","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17371873","citation_count":24,"is_preprint":false},{"pmid":"8700515","id":"PMC_8700515","title":"RN-tre identifies a family of tre-related proteins displaying a novel potential protein binding domain.","date":"1996","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/8700515","citation_count":22,"is_preprint":false},{"pmid":"35884836","id":"PMC_35884836","title":"Ubiquitin-Specific Protease 6 n-Terminal-like Protein (USP6NL) and the Epidermal Growth Factor Receptor (EGFR) Signaling Axis Regulates Ubiquitin-Mediated DNA Repair and Temozolomide-Resistance in Glioblastoma.","date":"2022","source":"Biomedicines","url":"https://pubmed.ncbi.nlm.nih.gov/35884836","citation_count":12,"is_preprint":false},{"pmid":"33054738","id":"PMC_33054738","title":"USP6NL mediated by LINC00689/miR-142-3p promotes the development of triple-negative breast cancer.","date":"2020","source":"BMC cancer","url":"https://pubmed.ncbi.nlm.nih.gov/33054738","citation_count":9,"is_preprint":false},{"pmid":"40643716","id":"PMC_40643716","title":"USP6NL knockdown suppresses colorectal cancer progression by inducing CASP9-Mediated apoptosis and disrupting FOXC2/SNAI1-Driven EMT and angiogenesis.","date":"2025","source":"Functional & integrative genomics","url":"https://pubmed.ncbi.nlm.nih.gov/40643716","citation_count":3,"is_preprint":false},{"pmid":"32816624","id":"PMC_32816624","title":"The Drosophila melanogaster Rab GAP RN-tre cross-talks with the Rho1 signaling pathway to regulate nonmuscle myosin II localization and function.","date":"2020","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/32816624","citation_count":3,"is_preprint":false},{"pmid":"40834681","id":"PMC_40834681","title":"USP6NL and MSX2 as the novel diagnostic markers in gastric cancer patients.","date":"2025","source":"Cancer treatment and research communications","url":"https://pubmed.ncbi.nlm.nih.gov/40834681","citation_count":0,"is_preprint":false},{"pmid":"42239457","id":"PMC_42239457","title":"A screen for adherens junction proteins regulating collective cell migration and testis morphogenesis reveals important roles for the Rab GAP RN-tre and the kinase Par-1.","date":"2026","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/42239457","citation_count":0,"is_preprint":false},{"pmid":"41401861","id":"PMC_41401861","title":"Molecular basis of Rab43 inactivation by RN-Tre in endocytic trafficking unveils a general Rab-GAP recognition mechanism.","date":"2025","source":"International journal of biological macromolecules","url":"https://pubmed.ncbi.nlm.nih.gov/41401861","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.04.07.25325386","title":"Silencer variants are key drivers of gene upregulation in Alzheimer’s disease","date":"2025-04-08","source":"bioRxiv","url":"https://doi.org/10.1101/2025.04.07.25325386","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10721,"output_tokens":2586,"usd":0.035477,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9702,"output_tokens":3080,"usd":0.062755,"stage2_stop_reason":"end_turn"},"total_usd":0.098232,"stage1_batch_id":"msgbatch_01UT1AahFPCAyn2uVweS7A9z","stage2_batch_id":"msgbatch_01HuKWtKNT4Pqj1bc8eEP5RV","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1996,\n      \"finding\": \"RN-tre (USP6NL) was identified as an 828 amino acid protein that binds specifically to the SH3 domain of Eps8 with high affinity (Kd 10^-8 to 10^-7 M), both in vitro and in vivo; the TrH (Tre Homology) domain in its N-terminus has protein-binding properties in vitro. A C-terminal truncated mutant conferred proliferative advantage and reduced serum requirement to NIH3T3 fibroblasts.\",\n      \"method\": \"In vitro binding assays, co-immunoprecipitation, NIH3T3 transformation assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal in vitro and in vivo binding with quantified Kd, functional truncation mutant, single lab\",\n      \"pmids\": [\"8700527\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"RN-tre (USP6NL) maps to chromosomal region 10p13, is ubiquitously expressed, and the tre oncogene was shown to be a fusion product of a 5' element homologous to RN-tre and a 3' element encoding a deubiquitinating enzyme; the TrH domain is conserved from yeast to mammals and has protein-binding properties in vitro.\",\n      \"method\": \"cDNA cloning, chromosomal mapping, in vitro protein binding assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct experimental mapping and domain characterization, single lab, two methods\",\n      \"pmids\": [\"8700515\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"RN-tre (USP6NL) associates with the Grb2 adaptor protein via Grb2's SH3 domains binding to proline-rich sequences in RN-tre, both in vitro and in vivo; this interaction is constitutive and independent of Eps8. EGF stimulates Grb2-dependent recruitment of RN-tre to the EGFR. Overexpression of RN-tre blocks internalization of EGFR, and a Grb2 mutant deficient in RN-tre binding is not blocked by RN-tre overexpression, demonstrating that RN-tre inhibits EGFR endocytosis through Grb2-mediated receptor binding.\",\n      \"method\": \"Co-immunoprecipitation (in vitro and in vivo), overexpression/dominant-negative assays, fluorescence microscopy\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP in vitro and in vivo, mechanistic dissection with Grb2 binding-deficient mutant, replicated across multiple experiments\",\n      \"pmids\": [\"12399475\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"RN-tre (USP6NL) is a physiological substrate of the human Cdc14A dual-specificity phosphatase. RN-tre undergoes cell cycle-dependent phosphorylation peaking at mitosis, phosphorylated by cyclin-dependent kinase; hCdc14A dephosphorylates RN-tre both in vitro and in vivo. RN-tre phosphorylation is required for efficient hCdc14A binding and finely modulates RN-tre's GAP catalytic activity.\",\n      \"method\": \"Genetic substrate-trapping (catalytically inactive hCdc14A C278S mutant), Co-immunoprecipitation, in vitro phosphatase assay, cell cycle synchronization, CDK phosphorylation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — substrate-trapping combined with in vitro phosphatase assay and in vivo validation, multiple orthogonal methods in a single rigorous study\",\n      \"pmids\": [\"17371873\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"RN-tre (USP6NL) localizes to focal adhesions and Rab5-positive vesicles associated with focal adhesions undergoing rapid remodeling. RN-tre acts as a GAP for Rab5 and Rab43 and inhibits endocytosis of β1 (but not β3) integrins, delaying focal adhesion turnover and impairing β1-dependent chemotactic cell migration. All effects require GAP activity and are Rab5-dependent.\",\n      \"method\": \"Live-cell imaging, loss-of-function (siRNA), rescue with GAP-dead mutant, integrin endocytosis assay, chemotaxis assay, fluorescence microscopy\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (live imaging, loss-of-function, GAP-dead mutant rescue, functional migration assay), integrin selectivity established\",\n      \"pmids\": [\"24239119\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"High USP6NL (RN-tre) levels in breast cancer cells delay endocytosis and degradation of EGFR, causing chronic AKT activation. In turn, activated AKT stabilizes GLUT1 at the plasma membrane, increasing aerobic glycolysis. Depletion of USP6NL accelerated EGFR/AKT downregulation and GLUT1 degradation, impairing proliferation specifically in USP6NL-high cancer cells.\",\n      \"method\": \"siRNA knockdown, EGFR endocytosis assay, western blot (phospho-AKT), GLUT1 membrane fractionation, glucose uptake assay, cell proliferation assay\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods establishing the USP6NL→EGFR→AKT→GLUT1 axis with knockdown and functional metabolic readouts\",\n      \"pmids\": [\"29691252\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"In Drosophila S2 cells, depletion of RN-tre (ortholog of USP6NL) leads to a punctate non-muscle myosin II (NMII) RLC phenotype, decreased active Rho1, and decreased phosphomyosin; constitutively active Rho or Rho-kinase (Rok) rescues this phenotype. RN-tre regulation of NMII is only partially dependent on GAP activity (a GAP-dead RN-tre partially restores phosphomyosin, and constitutively active Rab substrates do not recapitulate the NMII phenotype), suggesting RN-tre links the secretion/endocytic machinery to actomyosin contractility via Rho1 signaling.\",\n      \"method\": \"RNAi screen in Drosophila S2 cells, immunostaining (phosphomyosin), GAP-dead mutant, constitutively active Rho/Rok rescue, actin retrograde flow assay, contractility assay\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple methods (RNAi, mutant rescue, functional assays) in Drosophila ortholog, single lab\",\n      \"pmids\": [\"32816624\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The crystal structure of the RN-Tre (USP6NL)–Rab43 complex reveals a bipartite recognition mechanism: the N-terminal TBC subdomain catalytically remodels Rab43 Switch regions (via RQ-dual finger mechanism), while the C-terminal subdomain engages Switch II and reorients the hydrophobic triad to confer substrate specificity. Leu146 and C-terminal residues are key specificity determinants, and the same structural analysis identified Rab19 as an additional substrate. Disease-associated RN-Tre mutations impair GAP activity, resulting in aberrant Golgi architecture and endocytic trafficking.\",\n      \"method\": \"X-ray crystallography (crystal structure of RN-Tre–Rab43 complex), mutational analysis, in vitro GAP activity assay, functional assays for Golgi morphology and endocytic trafficking\",\n      \"journal\": \"International journal of biological macromolecules\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with mutagenesis and in vitro functional validation in a single rigorous study\",\n      \"pmids\": [\"41401861\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"USP6NL (RN-tre) is a TBC-domain Rab-GTPase-activating protein (GAP) that inactivates Rab5 and Rab43 (and Rab19) via a bipartite RQ-dual-finger mechanism structurally defined at atomic resolution; it is recruited to activated EGFR via constitutive association with Grb2, where it negatively regulates Rab5-dependent endocytosis, thereby prolonging EGFR/AKT signaling and stabilizing GLUT1 at the plasma membrane to fuel glycolysis; at focal adhesions it selectively inhibits β1-integrin endocytosis in a Rab5-GAP-dependent manner to control focal adhesion turnover and chemotactic migration; its GAP activity and cellular localization are regulated by CDK-dependent mitotic phosphorylation that is reversed by the hCdc14A phosphatase; and it also cross-talks with the Rho1–ROCK–myosin II contractility axis partly independently of its GAP activity.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"USP6NL (RN-tre) is a TBC-domain Rab-GTPase-activating protein that controls membrane trafficking by inactivating specific Rab GTPases, and through this activity it tunes receptor signaling, focal adhesion dynamics, and Golgi architecture [#2, #4, #7]. Structurally, it engages its substrate Rab43 through a bipartite recognition mechanism in which an N-terminal TBC subdomain catalytically remodels the Rab Switch regions via an RQ-dual-finger arrangement while a C-terminal subdomain enforces substrate specificity; this analysis defined Rab19 as an additional substrate and showed that disease-associated mutations impair GAP activity to produce aberrant Golgi architecture and endocytic trafficking [#7]. USP6NL acts as a GAP for Rab5 and Rab43 [#4]. It is recruited to activated EGFR through a constitutive, Eps8-independent association with the Grb2 adaptor, where it inhibits Rab5-dependent EGFR endocytosis; in breast cancer cells with high USP6NL this delays EGFR/AKT downregulation, stabilizes GLUT1 at the plasma membrane, and sustains aerobic glycolysis and proliferation [#2, #5]. At focal adhesions and associated Rab5-positive vesicles, USP6NL selectively blocks endocytosis of β1 (but not β3) integrins in a GAP- and Rab5-dependent manner, delaying focal adhesion turnover and impairing chemotactic migration [#4]. Its GAP activity is cell-cycle regulated through CDK-dependent mitotic phosphorylation that is reversed by the hCdc14A dual-specificity phosphatase [#3], and in Drosophila it additionally links the trafficking machinery to Rho1–ROCK–myosin II contractility partly independently of its GAP activity [#6].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Establishing USP6NL's molecular identity and first protein partner addressed what this newly cloned protein is and how it might act, revealing an Eps8-binding protein with transforming potential and a conserved TrH domain.\",\n      \"evidence\": \"cDNA cloning, chromosomal mapping, in vitro/in vivo SH3-binding assays, and NIH3T3 transformation assay\",\n      \"pmids\": [\"8700527\", \"8700515\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No enzymatic activity or substrate defined at this stage\", \"Mechanism of transformation by the truncation mutant unresolved\", \"Cellular pathway context unknown\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Identifying a constitutive Grb2 association resolved how USP6NL is targeted to its site of action, showing EGF-driven Grb2-dependent recruitment to EGFR that blocks receptor internalization.\",\n      \"evidence\": \"Reciprocal Co-IP in vitro and in vivo, Grb2 binding-deficient mutant, overexpression/dominant-negative assays, fluorescence microscopy\",\n      \"pmids\": [\"12399475\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the Rab substrate underlying the endocytic block\", \"Physiological versus overexpression-dependent effects not separated\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Demonstrating cell-cycle phosphorylation and hCdc14A-mediated dephosphorylation answered how USP6NL's catalytic activity is temporally regulated, linking its GAP function to mitotic control.\",\n      \"evidence\": \"Substrate-trapping with catalytically inactive hCdc14A, in vitro phosphatase assay, CDK phosphorylation assay, cell cycle synchronization, Co-IP\",\n      \"pmids\": [\"17371873\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific phosphosites and their individual functional consequences not fully mapped\", \"Trafficking consequence of mitotic regulation not directly tested\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defining USP6NL as a Rab5/Rab43 GAP at focal adhesions established its substrate specificity and a concrete cellular role, showing selective inhibition of β1-integrin endocytosis controls focal adhesion turnover and migration.\",\n      \"evidence\": \"Live-cell imaging, siRNA loss-of-function, GAP-dead rescue, integrin endocytosis and chemotaxis assays\",\n      \"pmids\": [\"24239119\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Basis for β1 versus β3 integrin selectivity not structurally explained\", \"Whether Grb2 recruitment operates at focal adhesions not addressed\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Connecting USP6NL to a EGFR→AKT→GLUT1 metabolic axis explained how its endocytic control feeds tumor metabolism, identifying it as a driver of aerobic glycolysis and proliferation in USP6NL-high breast cancer.\",\n      \"evidence\": \"siRNA knockdown, EGFR endocytosis assay, phospho-AKT western blot, GLUT1 membrane fractionation, glucose uptake and proliferation assays\",\n      \"pmids\": [\"29691252\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct GAP-dependence of the metabolic phenotype not isolated with a GAP-dead mutant\", \"In vivo tumor relevance not established here\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"An RNAi screen in Drosophila revealed a GAP-partially-independent link between USP6NL and Rho1–ROCK–myosin II contractility, expanding its role beyond canonical Rab inactivation.\",\n      \"evidence\": \"RNAi in S2 cells, phosphomyosin immunostaining, GAP-dead mutant, constitutively active Rho/Rok rescue, contractility and actin flow assays\",\n      \"pmids\": [\"32816624\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"GAP-independent mechanism on Rho1 not molecularly defined\", \"Conservation of this function in mammalian cells not confirmed\", \"Single lab, ortholog-based\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"The RN-Tre–Rab43 crystal structure resolved the long-open question of how USP6NL achieves catalysis and substrate selectivity, defining a bipartite RQ-dual-finger mechanism and linking disease mutations to impaired GAP activity and Golgi/endocytic defects.\",\n      \"evidence\": \"X-ray crystallography of the RN-Tre–Rab43 complex, mutational analysis, in vitro GAP assays, Golgi morphology and trafficking assays\",\n      \"pmids\": [\"41401861\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for Rab5 recognition not directly solved\", \"Specific disease and inheritance pattern not detailed in this analysis\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How USP6NL's distinct functional contexts — EGFR/Grb2 signaling, focal-adhesion integrin trafficking, mitotic phosphoregulation, Golgi maintenance, and Rho1-contractility crosstalk — are coordinated by a single GAP within one cell remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model integrating its multiple substrates and localizations\", \"Mechanism switching GAP-dependent versus GAP-independent functions unknown\", \"In vivo physiological hierarchy of these roles undetermined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [4, 7]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [7]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2, 5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [2, 4]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 5]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"GRB2\", \"EPS8\", \"CDC14A\", \"RAB5A\", \"RAB43\", \"EGFR\"],\n    \"other_free_text\": []\n  }\n}\n```","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}