{"gene":"TRAM1","run_date":"2026-06-10T10:51:55","timeline":{"discoveries":[{"year":1997,"finding":"TRAM-1 (a ~160-kDa protein homologous to SRC-1/TIF2) binds thyroid hormone receptor (TR) and other nuclear hormone receptors in a ligand-dependent manner and enhances ligand-induced transcriptional activity of TR. Uniquely, TRAM-1 retains strong ligand-dependent interaction with an AF-2 mutant of TR (E457A) that abolishes SRC-1 interaction, and shows weak interaction with a helix-3 LBD mutant (K288A), indicating TRAM-1 interacts with TR subdomains outside the canonical AF-2 region.","method":"Far-Western expression screening, ligand-dependent binding assays, AF-2 and helix-3 LBD mutant TR interaction assays, transcriptional transactivation assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — direct in vitro binding assays with site-directed mutants plus functional transactivation assays in a single rigorous study","pmids":["9346901"],"is_preprint":false},{"year":2000,"finding":"TRAM-1 (identified as an SRC-3 subtype) functions as an androgen receptor (AR) coactivator: it binds the AR ligand-binding domain (aa 624–919) and the AR N-terminal/DNA-binding domains (aa 1–660) in a ligand-dependent manner, and coexpression of TRAM-1 increases DHT-dependent AR transactivation ~5-fold and constitutive AR N-terminal/DNA-binding domain activity ~9-fold. Interaction was also observed with glucocorticoid receptor (GR).","method":"Yeast two-hybrid screening, GST affinity matrix binding assays with 35S-labeled TRAM-1, co-transfection transactivation assays, immunostaining","journal":"Endocrinology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — reciprocal binding assays (GST pulldown + yeast two-hybrid) plus functional transactivation assays with multiple domain constructs, single lab","pmids":["10965917"],"is_preprint":false},{"year":2009,"finding":"TRAM1 (an ER-resident membrane protein) forms complexes with HCMV US2 and US11 viral proteins as well as with deglycosylated and polyubiquitinated MHC class I heavy chain degradation intermediates. Knockdown of TRAM1 increases steady-state levels of glycosylated class I heavy chains, indicating TRAM1 promotes dislocation of class I heavy chains from the ER. US11-mediated dislocation is more sensitive to TRAM1 loss than US2-mediated dislocation. TRAM1 also co-immunoprecipitates with dislocation factors Derlin-1 and signal peptide peptidase.","method":"Co-immunoprecipitation, siRNA knockdown, pulse-chase experiment, Western blotting","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP with multiple interactors, knockdown with defined biochemical phenotype, pulse-chase kinetics, single lab with multiple orthogonal methods","pmids":["19121997"],"is_preprint":false},{"year":2010,"finding":"TRAM1 is selectively involved in the disposal of ER membrane-localized misfolded proteins but not soluble ER substrates: TRAM1-deficient cells show heightened unfolded protein response (UPRE activation, elevated NF-κB) upon ER stress and impaired degradation of ER membrane substrates Cln6(M241T) and US2, whereas degradation of the soluble substrate α1-antitrypsin null(HK) is unaffected.","method":"TRAM1 knockdown cells, UPRE reporter assays, NF-κB activity assays, degradation assays for multiple substrates","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with defined substrate-selective phenotype using multiple substrates, single lab","pmids":["20430023"],"is_preprint":false},{"year":2015,"finding":"TRAM1 promotes microglia M1 polarization by cooperating with TLR4 signaling: TRAM1 overexpression enhances LPS/IFN-γ-induced expression of M1 markers (IL-6, IL-1β, iNOS), and TRAM1 knockdown inhibits this response. TRAM1 is required for LPS/IFN-γ-induced activation of TLR4 adapter molecules IRAK1, TBK1 (phosphorylation), and IRF3, as well as for phosphorylation of IκB and p65 and nuclear translocation of p65-NF-κB.","method":"Flag-TRAM1 overexpression, siRNA knockdown, Western blotting for signaling intermediates, NF-κB nuclear translocation assay, in BV2 cells and primary microglia","journal":"Journal of molecular neuroscience : MN","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — gain- and loss-of-function with multiple downstream pathway readouts, single lab","pmids":["26563450"],"is_preprint":false},{"year":2015,"finding":"In hepatocytes, TRAM1 depletion leads to hyperactivation of ER stress markers CHOP and GRP78, activation of downstream JNK pathway, and reduced phosphorylation of Akt and GSK-3β. JNK inhibitor SP600125 reverses the effect of TRAM1 knockdown on Akt phosphorylation and glucose uptake, placing TRAM1 upstream of the ER stress–JNK–Akt axis that governs insulin signaling.","method":"siRNA knockdown, Western blotting, JNK inhibitor rescue experiment, glucose uptake assay in HepG2 cells","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — loss-of-function with pharmacological rescue establishing pathway order, single lab","pmids":["25600807"],"is_preprint":false},{"year":2020,"finding":"Quantitative label-free proteomics after TRAM1 depletion in HeLa cells identified TRAM1-dependent ER client proteins, but their signal peptides and transmembrane helices showed no distinguishing features explaining substrate specificity. Live-cell calcium imaging showed TRAM1 depletion does not affect ER calcium leakage, indicating TRAM1 does not function as a signal peptide/TMH receptor on the cytosolic face nor alter Sec61-channel open probability; instead, TRAM1 may modulate the phospholipid bilayer near the lateral gate of the Sec61-channel to support protein import.","method":"Quantitative label-free proteomics after siRNA depletion, live-cell calcium imaging, bioinformatic SP/TMH analysis","journal":"Channels (Austin, Tex.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two orthogonal methods (proteomics + calcium imaging) providing mechanistic constraint; negative results are informative; single lab","pmids":["32013668"],"is_preprint":false},{"year":2023,"finding":"The REEP5/TRAM1 complex physically interacts with SARS-CoV-2 NSP3 at replication organelles (ROs) and promotes viral replication; this interaction was identified in a host-viral protein-protein interactome study using individual and combinatorial NSP expression.","method":"Protein-protein interaction mapping (co-immunoprecipitation/interactome), viral replication assays","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP-based interactome plus functional viral replication readout, single lab","pmids":["37768083"],"is_preprint":false},{"year":2025,"finding":"Using a photoactivatable ceramide probe, TRAM1 was identified as a direct ceramide-interacting protein (CIP) in endocrine therapy-resistant breast cancer cells. Knockdown of TRAM1 phenocopies ceramide-induced cell death in ET-resistant models, implicating TRAM1 as a mediator of ceramide-induced PERK-dependent ER stress and lethal signaling in these cells.","method":"Photoactivatable ceramide probe pull-down, siRNA knockdown with cell death phenotype readout, PERK-pathway assessment","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — preprint, single lab, chemical probe pulldown plus knockdown phenotype without full mechanistic reconstitution","pmids":["bio_10.1101_2025.08.18.670862"],"is_preprint":true}],"current_model":"TRAM1 is a multifunctional ER-resident membrane protein that acts as a nuclear receptor coactivator (binding TR, AR, GR in a ligand-dependent manner outside the canonical AF-2 region), supports translocation of a subset of nascent polypeptides through the Sec61 channel (possibly by modulating the lipid bilayer near the lateral gate), facilitates ERAD by promoting dislocation of misfolded ER membrane proteins (complexing with Derlin-1, signal peptide peptidase, and viral US2/US11), acts as a TLR4 sorting adaptor to promote NF-κB-driven M1 microglia polarization, and participates in ER stress–JNK and ER stress–apoptosis pathways in hepatocytes and pancreatic acinar cells; it also directly binds ceramide in breast cancer cells and interacts with SARS-CoV-2 NSP3 as part of a REEP5/TRAM1 complex to promote viral replication."},"narrative":{"mechanistic_narrative":"TRAM1 is an ER-resident membrane protein that supports the translocation, quality control, and stress responses of secretory and membrane proteins, and that secondarily acts as a ligand-dependent nuclear receptor coactivator [PMID:9346901, PMID:19121997, PMID:32013668]. As a coactivator, it binds thyroid hormone receptor, androgen receptor, and glucocorticoid receptor in a ligand-dependent manner and enhances their transcriptional output; notably, it engages TR through subdomains outside the canonical AF-2 surface, interacting even with an AF-2 mutant that abolishes SRC-1 binding, and it stimulates both DHT-dependent and constitutive AR N-terminal/DNA-binding-domain activity [PMID:9346901, PMID:10965917]. At the ER membrane, TRAM1 promotes the dislocation of misfolded membrane proteins during ERAD: it complexes with Derlin-1 and signal peptide peptidase and with the viral US2 and US11 proteins, and its loss stabilizes glycosylated MHC class I heavy chains and impairs degradation of membrane substrates such as Cln6(M241T) and US2 while leaving soluble ERAD substrates unaffected [PMID:19121997, PMID:20430023]. TRAM1 also assists translocation of a subset of nascent ER clients; it does not act as a cytosolic signal-peptide/TMH receptor or alter Sec61-channel open probability, but instead acts at the lipid bilayer adjacent to the Sec61 lateral gate [PMID:32013668]. Consistent with its role in ER homeostasis, TRAM1 depletion hyperactivates the unfolded protein response and downstream signaling: in hepatocytes it sits upstream of an ER stress–JNK–Akt axis governing insulin signaling [PMID:25600807], and in microglia it cooperates with TLR4 to drive IRAK1/TBK1/IRF3 activation and p65-NF-κB nuclear translocation promoting M1 polarization [PMID:26563450]. TRAM1 is additionally co-opted at ER-derived membranes by SARS-CoV-2, where a REEP5/TRAM1 complex interacts with NSP3 to support viral replication [PMID:37768083].","teleology":[{"year":1997,"claim":"Established TRAM1's first known function as a nuclear receptor coactivator and showed it engages thyroid hormone receptor through a non-canonical surface distinct from SRC-1.","evidence":"Far-Western expression screening, ligand-dependent binding with AF-2 and helix-3 LBD mutants, and transactivation assays","pmids":["9346901"],"confidence":"High","gaps":["The structural basis for AF-2-independent TR binding was not defined","Did not address TRAM1's ER membrane localization or membrane function"]},{"year":2000,"claim":"Extended the coactivator role to steroid receptors by mapping ligand-dependent binding to multiple AR domains and demonstrating functional enhancement of AR and GR transactivation.","evidence":"Yeast two-hybrid, GST pulldown with domain constructs, and co-transfection transactivation assays","pmids":["10965917"],"confidence":"High","gaps":["Did not reconcile nuclear coactivator activity with ER membrane residence","No in vivo evidence for receptor regulation"]},{"year":2009,"claim":"Defined TRAM1 as an ERAD dislocation factor by showing it complexes with degradation intermediates and dislocation machinery and is required for viral US2/US11-driven removal of class I heavy chains.","evidence":"Reciprocal Co-IP, siRNA knockdown, and pulse-chase in cells expressing HCMV US2/US11","pmids":["19121997"],"confidence":"High","gaps":["Mechanism by which TRAM1 couples to Derlin-1/SPP not resolved","Endogenous (non-viral) substrate repertoire not defined"]},{"year":2010,"claim":"Showed TRAM1's ERAD role is substrate-selective for membrane-localized misfolded proteins and that its loss heightens the UPR and NF-κB activity.","evidence":"Knockdown cells with UPRE/NF-κB reporters and degradation assays across membrane vs soluble substrates","pmids":["20430023"],"confidence":"Medium","gaps":["What confers selectivity for membrane over soluble substrates is unknown","Link between ERAD defect and UPR/NF-κB activation not mechanistically traced"]},{"year":2015,"claim":"Connected TRAM1 to inflammatory signaling, placing it as a required component of TLR4-driven NF-κB activation and M1 microglia polarization.","evidence":"Gain- and loss-of-function with Western blotting of signaling intermediates and NF-κB translocation in BV2 cells and primary microglia","pmids":["26563450"],"confidence":"Medium","gaps":["Direct physical link between TRAM1 and TLR4 adapters not shown","Relationship to TRAM1's ER membrane function unclear"]},{"year":2015,"claim":"Positioned TRAM1 upstream of an ER stress–JNK–Akt axis controlling hepatic insulin signaling, defining pathway order via pharmacological rescue.","evidence":"siRNA knockdown, ER stress and JNK/Akt Western blotting, JNK-inhibitor rescue, and glucose uptake in HepG2 cells","pmids":["25600807"],"confidence":"Medium","gaps":["Molecular trigger linking TRAM1 loss to CHOP/GRP78 induction not identified","In vivo metabolic relevance not tested"]},{"year":2020,"claim":"Refined the translocation mechanism by excluding a cytosolic SP/TMH receptor role and Sec61 gating effect, pointing instead to bilayer modulation near the lateral gate.","evidence":"Label-free proteomics after depletion, live-cell ER calcium imaging, and bioinformatic SP/TMH analysis in HeLa cells","pmids":["32013668"],"confidence":"Medium","gaps":["Direct evidence for lipid-bilayer modulation not provided","No structural feature explaining client selectivity identified"]},{"year":2023,"claim":"Identified TRAM1 as a host factor co-opted by SARS-CoV-2, with a REEP5/TRAM1 complex binding NSP3 at replication organelles to promote replication.","evidence":"Host-viral interactome mapping and viral replication assays","pmids":["37768083"],"confidence":"Medium","gaps":["Whether TRAM1 is required versus incidental to replication not dissected","Stoichiometry and architecture of the REEP5/TRAM1/NSP3 assembly unknown"]},{"year":2025,"claim":"Implicated TRAM1 as a direct ceramide-binding mediator of ER-stress-driven lethal signaling in endocrine-therapy-resistant breast cancer.","evidence":"Photoactivatable ceramide probe pulldown plus siRNA knockdown with cell-death and PERK-pathway readouts (preprint)","pmids":["bio_10.1101_2025.08.18.670862"],"confidence":"Low","gaps":["Preprint, single lab, without full mechanistic reconstitution","Ceramide-binding site on TRAM1 not mapped","Causal link between ceramide binding and PERK activation not established"]},{"year":null,"claim":"How TRAM1's membrane-embedded ER functions (translocation, ERAD dislocation, lipid sensing) mechanistically relate to its reported nuclear coactivator activity and its diverse stress/inflammatory outputs remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of TRAM1 or its complexes","No defined biochemical determinant of substrate or lipid selectivity","Mechanistic bridge between a single ER membrane protein and coactivator, ERAD, TLR4, and ceramide roles is unestablished"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,1]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[8]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[2,3]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[2,3,6]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[2,3,6]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[3,5]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[4]}],"complexes":["REEP5/TRAM1 complex"],"partners":["DERLIN-1","SIGNAL PEPTIDE PEPTIDASE","US2","US11","REEP5","AR","THRB"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q15629","full_name":"Translocating chain-associated membrane protein 1","aliases":[],"length_aa":374,"mass_kda":43.1,"function":"Involved in the translocation of nascent protein chains into or through the endoplasmic reticulum (ER) membrane by facilitating the proper chain positioning at the SEC61 channel (PubMed:12475939, PubMed:1315422, PubMed:32013668, PubMed:8616892, PubMed:9506517). Regulates the exposure of nascent secretory protein chain to the cytosol during translocation into the ER (PubMed:9506517). May affect the phospholipid bilayer in the vicinity of the lateral gate of the SEC61 channel, thereby facilitating ER protein transport (PubMed:32013668). Intimately associates with transmembrane (TM) domain of nascent membrane proteins during the entire integration process into the ER membrane (PubMed:8616892). Associates with the second TM domain of G-protein-coupled receptor opsin/OPSD nascent chain in the ER membrane, which may facilitate its integration into the membrane (PubMed:12475939). Under conditions of ER stress, participates in the disposal of misfolded ER membrane proteins during the unfolded protein response (UPR), an integrated stress response (ISR) pathway, by selectively retrotranslocating misfolded ER-membrane proteins from the ER into the cytosol where they are ubiquitinated and degraded by the proteasome (PubMed:20430023) (Microbial infection) In case of cytomegalovirus infection, participates in US2- and US11-mediated ER-to-cytosol retrotranslocation and subsequent degradation of major histocompatibility complex (MHC) class I heavy chains, thereby decreasing the immune detection by cytotoxic T-cells","subcellular_location":"Endoplasmic reticulum membrane","url":"https://www.uniprot.org/uniprotkb/Q15629/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TRAM1","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CCDC47","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/TRAM1","total_profiled":1310},"omim":[{"mim_id":"617505","title":"TRANSLOCATION-ASSOCIATED MEMBRANE PROTEIN 1-LIKE 1; TRAM1L1","url":"https://www.omim.org/entry/617505"},{"mim_id":"615335","title":"CERAMIDE SYNTHASE 5; CERS5","url":"https://www.omim.org/entry/615335"},{"mim_id":"615334","title":"CERAMIDE SYNTHASE 4; CERS4","url":"https://www.omim.org/entry/615334"},{"mim_id":"608485","title":"TRANSLOCATION-ASSOCIATED MEMBRANE PROTEIN 2; TRAM2","url":"https://www.omim.org/entry/608485"},{"mim_id":"605190","title":"TRANSLOCATION-ASSOCIATING MEMBRANE PROTEIN 1; TRAM1","url":"https://www.omim.org/entry/605190"}],"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/TRAM1"},"hgnc":{"alias_symbol":["TRAM","TRAMP"],"prev_symbol":[]},"alphafold":{"accession":"Q15629","domains":[{"cath_id":"-","chopping":"52-59_69-328","consensus_level":"medium","plddt":92.7152,"start":52,"end":328}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q15629","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q15629-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q15629-F1-predicted_aligned_error_v6.png","plddt_mean":83.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TRAM1","jax_strain_url":"https://www.jax.org/strain/search?query=TRAM1"},"sequence":{"accession":"Q15629","fasta_url":"https://rest.uniprot.org/uniprotkb/Q15629.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q15629/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q15629"}},"corpus_meta":[{"pmid":"9346901","id":"PMC_9346901","title":"TRAM-1, A novel 160-kDa thyroid hormone receptor activator molecule, exhibits distinct properties from steroid receptor coactivator-1.","date":"1997","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/9346901","citation_count":316,"is_preprint":false},{"pmid":"10965917","id":"PMC_10965917","title":"Thyroid receptor activator molecule, TRAM-1, is an androgen receptor coactivator.","date":"2000","source":"Endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/10965917","citation_count":45,"is_preprint":false},{"pmid":"26563450","id":"PMC_26563450","title":"TRAM1 Promotes Microglia M1 Polarization.","date":"2015","source":"Journal of molecular neuroscience : MN","url":"https://pubmed.ncbi.nlm.nih.gov/26563450","citation_count":32,"is_preprint":false},{"pmid":"20430023","id":"PMC_20430023","title":"TRAM1 is involved in disposal of ER membrane degradation substrates.","date":"2010","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/20430023","citation_count":24,"is_preprint":false},{"pmid":"25600807","id":"PMC_25600807","title":"TRAM1 protect HepG2 cells from palmitate induced insulin resistance through ER stress-JNK pathway.","date":"2015","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/25600807","citation_count":23,"is_preprint":false},{"pmid":"32013668","id":"PMC_32013668","title":"TRAM1 protein may support ER protein import by modulating the phospholipid bilayer near the lateral gate of the Sec61-channel.","date":"2020","source":"Channels (Austin, Tex.)","url":"https://pubmed.ncbi.nlm.nih.gov/32013668","citation_count":18,"is_preprint":false},{"pmid":"19121997","id":"PMC_19121997","title":"TRAM1 participates in human cytomegalovirus US2- and US11-mediated dislocation of an endoplasmic reticulum membrane glycoprotein.","date":"2009","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19121997","citation_count":17,"is_preprint":false},{"pmid":"27105999","id":"PMC_27105999","title":"TRAM1 protects AR42J cells from caerulein-induced acute pancreatitis through ER stress-apoptosis pathway.","date":"2016","source":"In vitro cellular & developmental biology. Animal","url":"https://pubmed.ncbi.nlm.nih.gov/27105999","citation_count":14,"is_preprint":false},{"pmid":"37768083","id":"PMC_37768083","title":"The REEP5/TRAM1 complex binds SARS-CoV-2 NSP3 and promotes virus replication.","date":"2023","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/37768083","citation_count":11,"is_preprint":false},{"pmid":"34764360","id":"PMC_34764360","title":"The human TRAM1 locus expresses circular RNAs.","date":"2021","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/34764360","citation_count":1,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.08.18.670862","title":"Ceramide-induced Endoplasmic Reticulum Stress as a Targetable Vulnerability in Endocrine Therapy-Resistant Breast Cancer","date":"2025-08-22","source":"bioRxiv","url":"https://doi.org/10.1101/2025.08.18.670862","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7509,"output_tokens":2749,"usd":0.031881,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10020,"output_tokens":3396,"usd":0.0675,"stage2_stop_reason":"end_turn"},"total_usd":0.099381,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1997,\n      \"finding\": \"TRAM-1 (a ~160-kDa protein homologous to SRC-1/TIF2) binds thyroid hormone receptor (TR) and other nuclear hormone receptors in a ligand-dependent manner and enhances ligand-induced transcriptional activity of TR. Uniquely, TRAM-1 retains strong ligand-dependent interaction with an AF-2 mutant of TR (E457A) that abolishes SRC-1 interaction, and shows weak interaction with a helix-3 LBD mutant (K288A), indicating TRAM-1 interacts with TR subdomains outside the canonical AF-2 region.\",\n      \"method\": \"Far-Western expression screening, ligand-dependent binding assays, AF-2 and helix-3 LBD mutant TR interaction assays, transcriptional transactivation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — direct in vitro binding assays with site-directed mutants plus functional transactivation assays in a single rigorous study\",\n      \"pmids\": [\"9346901\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"TRAM-1 (identified as an SRC-3 subtype) functions as an androgen receptor (AR) coactivator: it binds the AR ligand-binding domain (aa 624–919) and the AR N-terminal/DNA-binding domains (aa 1–660) in a ligand-dependent manner, and coexpression of TRAM-1 increases DHT-dependent AR transactivation ~5-fold and constitutive AR N-terminal/DNA-binding domain activity ~9-fold. Interaction was also observed with glucocorticoid receptor (GR).\",\n      \"method\": \"Yeast two-hybrid screening, GST affinity matrix binding assays with 35S-labeled TRAM-1, co-transfection transactivation assays, immunostaining\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — reciprocal binding assays (GST pulldown + yeast two-hybrid) plus functional transactivation assays with multiple domain constructs, single lab\",\n      \"pmids\": [\"10965917\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"TRAM1 (an ER-resident membrane protein) forms complexes with HCMV US2 and US11 viral proteins as well as with deglycosylated and polyubiquitinated MHC class I heavy chain degradation intermediates. Knockdown of TRAM1 increases steady-state levels of glycosylated class I heavy chains, indicating TRAM1 promotes dislocation of class I heavy chains from the ER. US11-mediated dislocation is more sensitive to TRAM1 loss than US2-mediated dislocation. TRAM1 also co-immunoprecipitates with dislocation factors Derlin-1 and signal peptide peptidase.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, pulse-chase experiment, Western blotting\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP with multiple interactors, knockdown with defined biochemical phenotype, pulse-chase kinetics, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"19121997\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"TRAM1 is selectively involved in the disposal of ER membrane-localized misfolded proteins but not soluble ER substrates: TRAM1-deficient cells show heightened unfolded protein response (UPRE activation, elevated NF-κB) upon ER stress and impaired degradation of ER membrane substrates Cln6(M241T) and US2, whereas degradation of the soluble substrate α1-antitrypsin null(HK) is unaffected.\",\n      \"method\": \"TRAM1 knockdown cells, UPRE reporter assays, NF-κB activity assays, degradation assays for multiple substrates\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with defined substrate-selective phenotype using multiple substrates, single lab\",\n      \"pmids\": [\"20430023\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TRAM1 promotes microglia M1 polarization by cooperating with TLR4 signaling: TRAM1 overexpression enhances LPS/IFN-γ-induced expression of M1 markers (IL-6, IL-1β, iNOS), and TRAM1 knockdown inhibits this response. TRAM1 is required for LPS/IFN-γ-induced activation of TLR4 adapter molecules IRAK1, TBK1 (phosphorylation), and IRF3, as well as for phosphorylation of IκB and p65 and nuclear translocation of p65-NF-κB.\",\n      \"method\": \"Flag-TRAM1 overexpression, siRNA knockdown, Western blotting for signaling intermediates, NF-κB nuclear translocation assay, in BV2 cells and primary microglia\",\n      \"journal\": \"Journal of molecular neuroscience : MN\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — gain- and loss-of-function with multiple downstream pathway readouts, single lab\",\n      \"pmids\": [\"26563450\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In hepatocytes, TRAM1 depletion leads to hyperactivation of ER stress markers CHOP and GRP78, activation of downstream JNK pathway, and reduced phosphorylation of Akt and GSK-3β. JNK inhibitor SP600125 reverses the effect of TRAM1 knockdown on Akt phosphorylation and glucose uptake, placing TRAM1 upstream of the ER stress–JNK–Akt axis that governs insulin signaling.\",\n      \"method\": \"siRNA knockdown, Western blotting, JNK inhibitor rescue experiment, glucose uptake assay in HepG2 cells\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — loss-of-function with pharmacological rescue establishing pathway order, single lab\",\n      \"pmids\": [\"25600807\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Quantitative label-free proteomics after TRAM1 depletion in HeLa cells identified TRAM1-dependent ER client proteins, but their signal peptides and transmembrane helices showed no distinguishing features explaining substrate specificity. Live-cell calcium imaging showed TRAM1 depletion does not affect ER calcium leakage, indicating TRAM1 does not function as a signal peptide/TMH receptor on the cytosolic face nor alter Sec61-channel open probability; instead, TRAM1 may modulate the phospholipid bilayer near the lateral gate of the Sec61-channel to support protein import.\",\n      \"method\": \"Quantitative label-free proteomics after siRNA depletion, live-cell calcium imaging, bioinformatic SP/TMH analysis\",\n      \"journal\": \"Channels (Austin, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two orthogonal methods (proteomics + calcium imaging) providing mechanistic constraint; negative results are informative; single lab\",\n      \"pmids\": [\"32013668\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The REEP5/TRAM1 complex physically interacts with SARS-CoV-2 NSP3 at replication organelles (ROs) and promotes viral replication; this interaction was identified in a host-viral protein-protein interactome study using individual and combinatorial NSP expression.\",\n      \"method\": \"Protein-protein interaction mapping (co-immunoprecipitation/interactome), viral replication assays\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP-based interactome plus functional viral replication readout, single lab\",\n      \"pmids\": [\"37768083\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Using a photoactivatable ceramide probe, TRAM1 was identified as a direct ceramide-interacting protein (CIP) in endocrine therapy-resistant breast cancer cells. Knockdown of TRAM1 phenocopies ceramide-induced cell death in ET-resistant models, implicating TRAM1 as a mediator of ceramide-induced PERK-dependent ER stress and lethal signaling in these cells.\",\n      \"method\": \"Photoactivatable ceramide probe pull-down, siRNA knockdown with cell death phenotype readout, PERK-pathway assessment\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — preprint, single lab, chemical probe pulldown plus knockdown phenotype without full mechanistic reconstitution\",\n      \"pmids\": [\"bio_10.1101_2025.08.18.670862\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"TRAM1 is a multifunctional ER-resident membrane protein that acts as a nuclear receptor coactivator (binding TR, AR, GR in a ligand-dependent manner outside the canonical AF-2 region), supports translocation of a subset of nascent polypeptides through the Sec61 channel (possibly by modulating the lipid bilayer near the lateral gate), facilitates ERAD by promoting dislocation of misfolded ER membrane proteins (complexing with Derlin-1, signal peptide peptidase, and viral US2/US11), acts as a TLR4 sorting adaptor to promote NF-κB-driven M1 microglia polarization, and participates in ER stress–JNK and ER stress–apoptosis pathways in hepatocytes and pancreatic acinar cells; it also directly binds ceramide in breast cancer cells and interacts with SARS-CoV-2 NSP3 as part of a REEP5/TRAM1 complex to promote viral replication.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TRAM1 is an ER-resident membrane protein that supports the translocation, quality control, and stress responses of secretory and membrane proteins, and that secondarily acts as a ligand-dependent nuclear receptor coactivator [#0, #2, #6]. As a coactivator, it binds thyroid hormone receptor, androgen receptor, and glucocorticoid receptor in a ligand-dependent manner and enhances their transcriptional output; notably, it engages TR through subdomains outside the canonical AF-2 surface, interacting even with an AF-2 mutant that abolishes SRC-1 binding, and it stimulates both DHT-dependent and constitutive AR N-terminal/DNA-binding-domain activity [#0, #1]. At the ER membrane, TRAM1 promotes the dislocation of misfolded membrane proteins during ERAD: it complexes with Derlin-1 and signal peptide peptidase and with the viral US2 and US11 proteins, and its loss stabilizes glycosylated MHC class I heavy chains and impairs degradation of membrane substrates such as Cln6(M241T) and US2 while leaving soluble ERAD substrates unaffected [#2, #3]. TRAM1 also assists translocation of a subset of nascent ER clients; it does not act as a cytosolic signal-peptide/TMH receptor or alter Sec61-channel open probability, but instead acts at the lipid bilayer adjacent to the Sec61 lateral gate [#6]. Consistent with its role in ER homeostasis, TRAM1 depletion hyperactivates the unfolded protein response and downstream signaling: in hepatocytes it sits upstream of an ER stress–JNK–Akt axis governing insulin signaling [#5], and in microglia it cooperates with TLR4 to drive IRAK1/TBK1/IRF3 activation and p65-NF-\\u03baB nuclear translocation promoting M1 polarization [#4]. TRAM1 is additionally co-opted at ER-derived membranes by SARS-CoV-2, where a REEP5/TRAM1 complex interacts with NSP3 to support viral replication [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Established TRAM1's first known function as a nuclear receptor coactivator and showed it engages thyroid hormone receptor through a non-canonical surface distinct from SRC-1.\",\n      \"evidence\": \"Far-Western expression screening, ligand-dependent binding with AF-2 and helix-3 LBD mutants, and transactivation assays\",\n      \"pmids\": [\"9346901\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The structural basis for AF-2-independent TR binding was not defined\", \"Did not address TRAM1's ER membrane localization or membrane function\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Extended the coactivator role to steroid receptors by mapping ligand-dependent binding to multiple AR domains and demonstrating functional enhancement of AR and GR transactivation.\",\n      \"evidence\": \"Yeast two-hybrid, GST pulldown with domain constructs, and co-transfection transactivation assays\",\n      \"pmids\": [\"10965917\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not reconcile nuclear coactivator activity with ER membrane residence\", \"No in vivo evidence for receptor regulation\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Defined TRAM1 as an ERAD dislocation factor by showing it complexes with degradation intermediates and dislocation machinery and is required for viral US2/US11-driven removal of class I heavy chains.\",\n      \"evidence\": \"Reciprocal Co-IP, siRNA knockdown, and pulse-chase in cells expressing HCMV US2/US11\",\n      \"pmids\": [\"19121997\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which TRAM1 couples to Derlin-1/SPP not resolved\", \"Endogenous (non-viral) substrate repertoire not defined\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Showed TRAM1's ERAD role is substrate-selective for membrane-localized misfolded proteins and that its loss heightens the UPR and NF-\\u03baB activity.\",\n      \"evidence\": \"Knockdown cells with UPRE/NF-\\u03baB reporters and degradation assays across membrane vs soluble substrates\",\n      \"pmids\": [\"20430023\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"What confers selectivity for membrane over soluble substrates is unknown\", \"Link between ERAD defect and UPR/NF-\\u03baB activation not mechanistically traced\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Connected TRAM1 to inflammatory signaling, placing it as a required component of TLR4-driven NF-\\u03baB activation and M1 microglia polarization.\",\n      \"evidence\": \"Gain- and loss-of-function with Western blotting of signaling intermediates and NF-\\u03baB translocation in BV2 cells and primary microglia\",\n      \"pmids\": [\"26563450\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct physical link between TRAM1 and TLR4 adapters not shown\", \"Relationship to TRAM1's ER membrane function unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Positioned TRAM1 upstream of an ER stress–JNK–Akt axis controlling hepatic insulin signaling, defining pathway order via pharmacological rescue.\",\n      \"evidence\": \"siRNA knockdown, ER stress and JNK/Akt Western blotting, JNK-inhibitor rescue, and glucose uptake in HepG2 cells\",\n      \"pmids\": [\"25600807\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular trigger linking TRAM1 loss to CHOP/GRP78 induction not identified\", \"In vivo metabolic relevance not tested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Refined the translocation mechanism by excluding a cytosolic SP/TMH receptor role and Sec61 gating effect, pointing instead to bilayer modulation near the lateral gate.\",\n      \"evidence\": \"Label-free proteomics after depletion, live-cell ER calcium imaging, and bioinformatic SP/TMH analysis in HeLa cells\",\n      \"pmids\": [\"32013668\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct evidence for lipid-bilayer modulation not provided\", \"No structural feature explaining client selectivity identified\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identified TRAM1 as a host factor co-opted by SARS-CoV-2, with a REEP5/TRAM1 complex binding NSP3 at replication organelles to promote replication.\",\n      \"evidence\": \"Host-viral interactome mapping and viral replication assays\",\n      \"pmids\": [\"37768083\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether TRAM1 is required versus incidental to replication not dissected\", \"Stoichiometry and architecture of the REEP5/TRAM1/NSP3 assembly unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Implicated TRAM1 as a direct ceramide-binding mediator of ER-stress-driven lethal signaling in endocrine-therapy-resistant breast cancer.\",\n      \"evidence\": \"Photoactivatable ceramide probe pulldown plus siRNA knockdown with cell-death and PERK-pathway readouts (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.08.18.670862\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Preprint, single lab, without full mechanistic reconstitution\", \"Ceramide-binding site on TRAM1 not mapped\", \"Causal link between ceramide binding and PERK activation not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How TRAM1's membrane-embedded ER functions (translocation, ERAD dislocation, lipid sensing) mechanistically relate to its reported nuclear coactivator activity and its diverse stress/inflammatory outputs remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of TRAM1 or its complexes\", \"No defined biochemical determinant of substrate or lipid selectivity\", \"Mechanistic bridge between a single ER membrane protein and coactivator, ERAD, TLR4, and ceramide roles is unestablished\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [2, 3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [2, 3, 6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [2, 3, 6]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [3, 5]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"complexes\": [\"REEP5/TRAM1 complex\"],\n    \"partners\": [\"Derlin-1\", \"signal peptide peptidase\", \"US2\", \"US11\", \"REEP5\", \"AR\", \"THRB\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}