{"gene":"RNF167","run_date":"2026-04-28T19:45:45","timeline":{"discoveries":[{"year":2012,"finding":"RNF167 is a transmembrane RING domain-containing E3 ubiquitin ligase that is predominantly lysosomal (with a subpopulation on the neuronal surface) and ubiquitinates AMPA receptor subunits in an activity-dependent manner, thereby reducing AMPAR surface expression and synaptic AMPAR currents without affecting NMDAR currents.","method":"shRNA knockdown, RING-dead mutant overexpression, surface expression assays, electrophysiology in hippocampal neurons, subcellular localization by fluorescence microscopy","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (genetic KD, dominant-negative mutant, electrophysiology, localization) in a single study","pmids":["23129617"],"is_preprint":false},{"year":2014,"finding":"Point mutations in the RING domain of RNF167 abrogate its E3 ubiquitin ligase activity, and the PA (protease-associated) domain is required for endosomal localization; PA domain mutations found in human tumors retain ligase activity but cause mislocalization, indicating both enzymatic activity and correct localization are needed for substrate targeting.","method":"Mutagenesis of RING and PA domains, ubiquitination assays, subcellular localization microscopy","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1/2 — in vitro ligase assays combined with mutagenesis and localization studies","pmids":["24387786"],"is_preprint":false},{"year":2016,"finding":"RNF167 ubiquitinates the small GTPase Arl8B at lysine K141, targeting it for degradation; RNF167 overexpression reduces Arl8B levels and alters lysosome positioning and endocytic trafficking to lysosomes, effects that are counteracted by the ubiquitination-defective Arl8B K141R mutant.","method":"Proximity-dependent biotin labeling (BioID) for substrate identification, in vitro ubiquitination assay, site-directed mutagenesis (K141R), overexpression/knockdown with lysosome positioning and endocytic trafficking readouts","journal":"The FEBS journal","confidence":"High","confidence_rationale":"Tier 1/2 — substrate identified by BioID, confirmed by in vitro ubiquitination and mutagenesis with defined cellular phenotype","pmids":["27808481"],"is_preprint":false},{"year":2021,"finding":"RNF167 ubiquitinates CASTOR1 with K29-linked polyubiquitin chains, leading to its proteasomal degradation; AKT phosphorylates CASTOR1 at S14, which increases CASTOR1 binding to RNF167 and promotes its ubiquitination/degradation while reducing its binding to MIOS, thereby activating mTORC1 in an arginine-independent manner.","method":"Co-immunoprecipitation, in vitro ubiquitination assays, ubiquitin linkage-specific analysis, phosphorylation assays, mTORC1 activity readouts, knockdown/knockout with xenograft tumor models","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1/2 — multiple orthogonal biochemical methods with in vivo functional validation","pmids":["33594058"],"is_preprint":false},{"year":2021,"finding":"RNF167 functionally interacts with E2 conjugating enzymes UBE2D1 and UBE2N; UBE2N drives K63-linked polyubiquitination of GluA2 (AMPAR subunit) only after GluA2 has been primed by a UBE2D1-initiated monoubiquitin, and pharmacological inhibition of UBE2N in hippocampal neurons reduces AMPA-induced GluA2 ubiquitination.","method":"In vitro autoubiquitination assays, binding assays with kinetic analysis, fluorescence microscopy co-localization, computational modeling of RING–E2 interaction, pharmacological inhibition of UBE2N in primary neurons","journal":"The FEBS journal","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution of polyubiquitination cascade with mechanistic dissection of E2 contributions, replicated in neurons","pmids":["33650289"],"is_preprint":false},{"year":2022,"finding":"RNF167 (E3) and deubiquitinase STAMBPL1 oppositely control polyubiquitination of Sestrin2; RNF167-mediated ubiquitination of Sestrin2 promotes its interaction with GATOR2 and inhibits mTORC1 signaling in response to leucine availability.","method":"Co-immunoprecipitation, ubiquitination assays, mTORC1 activity measurements, CRISPR knockout and heterozygous mutation correction, cell-permeable peptide blocking STAMBPL1-Sestrin2 interaction, xenograft tumor models","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1/2 — multiple orthogonal biochemical and cellular methods, in vivo tumor models, functional peptide validation","pmids":["35114100"],"is_preprint":false},{"year":2022,"finding":"RNF167 ubiquitinates Rab7 in a manner dependent on Rab7 being in its GTP-bound active (membrane-anchored) form; RNF167-mediated ubiquitination affects Rab7 membrane localization and results in larger Rab7-positive vesicles, and Charcot-Marie-Tooth Type 2B disease variants of Rab7 impair RNF167-mediated ubiquitination.","method":"Immunoprecipitation, in vitro ubiquitination assays, subcellular fractionation, epifluorescence microscopy, RNF167 knockdown with Lamp1 and vesicle size readouts","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 — multiple methods but single lab, no mutagenesis of ubiquitination sites on Rab7","pmids":["35887194"],"is_preprint":false},{"year":2023,"finding":"RNF167 interacts with Tollip via Tollip's CUE domain and attaches K33-linked polyubiquitin chains to Tollip at K235; this ubiquitination is required for Tollip to suppress TNF-α-induced NF-κB and MAPK (JNK) signaling, as the K235R Tollip mutant fails to inhibit these cascades.","method":"Co-immunoprecipitation, in vitro/cellular ubiquitination assays, ubiquitin linkage analysis, K235R point mutagenesis, NF-κB/MAPK reporter/kinase assays","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 — reciprocal Co-IP and mutagenesis with defined signaling phenotype, single lab","pmids":["37410058"],"is_preprint":false},{"year":2025,"finding":"RNF167 attaches atypical K6-linked polyubiquitin chains to the CARD domains of RIG-I and MDA5, marking them for p62-mediated selective autophagic degradation in autolysosomes; it also attaches K11-linked polyubiquitin chains to the CTD domains of RIG-I/MDA5, targeting them for proteasomal degradation. Both pathways synergistically suppress RLR-triggered type I interferon signaling.","method":"Co-immunoprecipitation, in vitro ubiquitination assays, ubiquitin linkage-specific analysis, domain-specific mutagenesis, autophagy flux assays (p62 interaction), proteasome inhibitor experiments, IFN-I reporter assays, RNF167 knockout","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1/2 — mechanistically dissects two distinct ubiquitin linkages on two distinct domains with orthogonal degradation pathway readouts","pmids":["39994288"],"is_preprint":false},{"year":2025,"finding":"RNF167 forms a heterodimer with the related E3 ubiquitin ligase RNF13, and this heterodimer modifies both RNF13 and RNF167 lysosomal trafficking; the heterodimer interacts with and alters IDS (iduronate 2-sulfatase) glycosylation and maturation differently than either protein alone, though RNF167's catalytic activity is not required for generating the underglycosylated IDS form.","method":"Co-immunoprecipitation, AlphaFold3 structural prediction (functional co-IP validation), catalytic mutant analysis, glycosylation and lysosomal trafficking assays","journal":"bioRxiv (preprint)","confidence":"Low","confidence_rationale":"Tier 3 — preprint, single Co-IP plus mutagenesis, no independent replication","pmids":["bio_10.1101_2025.06.20.660705"],"is_preprint":true},{"year":2025,"finding":"SMAD3 stabilizes Sestrin2 protein by modulating the balance between RNF167-mediated ubiquitination and STAMBPL1-mediated deubiquitination of Sestrin2, as shown by co-immunoprecipitation of SMAD3 with both enzymes.","method":"Co-immunoprecipitation, Western blotting, RT-qPCR, immunofluorescence, SMAD3 overexpression/knockdown","journal":"Cell division","confidence":"Low","confidence_rationale":"Tier 3 — single Co-IP without in vitro reconstitution; regulatory placement is correlative","pmids":["40751214"],"is_preprint":false}],"current_model":"RNF167 is a transmembrane RING-type E3 ubiquitin ligase that resides in endosomes and lysosomes (PA domain-dependent localization) and ubiquitinates a range of substrates—including AMPAR subunits (via UBE2D1/UBE2N), Arl8B, Rab7, CASTOR1, Sestrin2, Tollip, and RIG-I/MDA5—using distinct ubiquitin chain linkages (K6, K11, K29, K33, K63) to regulate lysosome positioning, endocytic trafficking, mTORC1 signaling, synaptic strength, NF-κB/MAPK signaling, and antiviral innate immune responses."},"narrative":{"teleology":[{"year":2012,"claim":"Establishing RNF167 as a lysosomal E3 ligase that controls synaptic AMPAR levels answered the question of whether endolysosomal ubiquitin ligases directly regulate ionotropic receptor trafficking at synapses.","evidence":"shRNA knockdown, RING-dead mutant, surface expression assays, and electrophysiology in hippocampal neurons","pmids":["23129617"],"confidence":"High","gaps":["Ubiquitination sites on AMPAR subunits not mapped","E2 conjugating enzymes not identified","Mechanism linking lysosomal RNF167 to surface receptor removal unclear"]},{"year":2014,"claim":"Demonstrating that the PA domain controls endosomal targeting while the RING domain provides catalytic activity resolved how RNF167 couples its enzymatic function to correct subcellular localization, and showed that cancer-associated PA mutations cause mislocalization without loss of ligase activity.","evidence":"Systematic mutagenesis of RING and PA domains with in vitro ubiquitination assays and subcellular localization microscopy","pmids":["24387786"],"confidence":"High","gaps":["Structural basis of PA domain–membrane interaction unknown","Whether PA-mutant mislocalization contributes to tumorigenesis not tested in vivo"]},{"year":2016,"claim":"Identifying Arl8B as a direct RNF167 substrate ubiquitinated at K141 established a mechanism by which RNF167 controls lysosome positioning and endocytic trafficking to lysosomes.","evidence":"BioID proximity labeling for substrate discovery, in vitro ubiquitination, K141R mutagenesis, lysosome positioning and trafficking assays","pmids":["27808481"],"confidence":"High","gaps":["Ubiquitin chain type on Arl8B not determined","Whether Arl8B degradation is proteasomal or lysosomal not resolved"]},{"year":2021,"claim":"Two studies revealed the E2 selectivity of RNF167 (UBE2D1 primes, UBE2N extends K63 chains on GluA2) and identified CASTOR1 as a substrate whose K29-linked ubiquitination by RNF167 integrates AKT phosphorylation with mTORC1 activation, answering how RNF167 builds specific ubiquitin chain types and connects to nutrient signaling.","evidence":"In vitro reconstitution of E2–E3 ubiquitination cascade with kinetic analysis and pharmacological UBE2N inhibition in neurons; Co-IP, ubiquitin linkage analysis, phosphorylation assays, and xenograft models for CASTOR1","pmids":["33650289","33594058"],"confidence":"High","gaps":["Whether UBE2D1/UBE2N pairing applies to non-AMPAR substrates unknown","Structural basis of RNF167–CASTOR1 recognition not determined"]},{"year":2022,"claim":"Discovery that RNF167 ubiquitinates Sestrin2 to promote GATOR2 interaction and suppress mTORC1 in response to leucine, and ubiquitinates GTP-bound Rab7 affecting late endosome morphology, broadened RNF167's role to nutrient-responsive mTORC1 inhibition and endosomal GTPase regulation.","evidence":"CRISPR knockout, cell-permeable peptide, xenograft models for Sestrin2; in vitro ubiquitination, subcellular fractionation, vesicle size analysis for Rab7","pmids":["35114100","35887194"],"confidence":"High","gaps":["Ubiquitin chain type on Rab7 not determined","Ubiquitination sites on Rab7 not mapped","How RNF167 exerts opposing effects on mTORC1 via CASTOR1 versus Sestrin2 is not integrated"]},{"year":2023,"claim":"Identification of Tollip as a substrate receiving K33-linked ubiquitin at K235, required for Tollip-mediated suppression of TNF-α-induced NF-κB and JNK signaling, expanded RNF167's repertoire to inflammatory signaling regulation via atypical chain types.","evidence":"Reciprocal Co-IP, ubiquitin linkage analysis, K235R mutagenesis, NF-κB/MAPK reporter assays","pmids":["37410058"],"confidence":"Medium","gaps":["Single-lab finding awaiting independent replication","Whether RNF167–Tollip axis operates in vivo during inflammation not tested","Mechanism of K33 chain recognition by downstream effectors unknown"]},{"year":2025,"claim":"Mechanistic dissection of dual ubiquitin chain types on RIG-I/MDA5 (K6-linked on CARD for autophagic degradation via p62; K11-linked on CTD for proteasomal degradation) revealed how RNF167 suppresses antiviral innate immunity through two synergistic degradation pathways.","evidence":"Domain-specific mutagenesis, linkage-specific ubiquitin analysis, autophagy flux assays, proteasome inhibitor experiments, IFN-I reporters, RNF167 knockout","pmids":["39994288"],"confidence":"High","gaps":["In vivo relevance during viral infection not demonstrated","How RNF167 selects K6 versus K11 linkages on different domains mechanistically unclear","E2 enzymes mediating K6 and K11 chain assembly not identified"]},{"year":null,"claim":"Key unresolved questions include how RNF167 coordinates opposing effects on mTORC1 (CASTOR1 degradation activates, Sestrin2 ubiquitination inhibits), the structural basis of PA domain-mediated membrane targeting, and whether the RNF167–RNF13 heterodimer represents a physiologically relevant complex with distinct substrate specificity.","evidence":"","pmids":[],"confidence":"High","gaps":["No structural model of full-length RNF167 or its PA domain–membrane interaction","Context-dependent regulation of RNF167 toward opposing mTORC1 outcomes not resolved","RNF167–RNF13 heterodimer function supported only by preprint data"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,2,3,4,5,6,7,8]}],"localization":[{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[0,1,2]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[1,6]}],"pathway":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,5,7]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,2,3,4,5,6,7,8]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[8]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[8]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[0,4]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[2,6]}],"complexes":[],"partners":["UBE2D1","UBE2N","ARL8B","RAB7A","CASTOR1","SESN2","TOLLIP","DDX58"],"other_free_text":[]},"mechanistic_narrative":"RNF167 is a transmembrane RING-type E3 ubiquitin ligase that resides on endosomes and lysosomes—dependent on its protease-associated (PA) domain—and ubiquitinates diverse substrates using atypical ubiquitin chain linkages to regulate endolysosomal trafficking, mTORC1 nutrient sensing, synaptic plasticity, and innate immune signaling. It ubiquitinates AMPA receptor subunits via a two-step mechanism in which UBE2D1 primes monoubiquitination and UBE2N extends K63-linked chains, reducing synaptic AMPAR surface expression [PMID:23129617, PMID:33650289]. RNF167 targets the lysosome-positioning GTPase Arl8B (K141) and GTP-bound Rab7 for ubiquitin-dependent degradation, controlling lysosome distribution and endocytic flux [PMID:27808481, PMID:35887194], and regulates mTORC1 through opposing ubiquitination events on CASTOR1 (K29-linked chains promoting proteasomal degradation to activate mTORC1) and Sestrin2 (polyubiquitination enhancing GATOR2 binding to inhibit mTORC1) [PMID:33594058, PMID:35114100]. It also attaches K6-linked chains to the CARD domains and K11-linked chains to the CTD domains of RIG-I and MDA5, directing them to autophagic and proteasomal degradation respectively, thereby suppressing type I interferon signaling [PMID:39994288]."},"prefetch_data":{"uniprot":{"accession":"Q9H6Y7","full_name":"E3 ubiquitin-protein ligase RNF167","aliases":["RING finger protein 167"],"length_aa":350,"mass_kda":38.3,"function":"E3 ubiquitin-protein ligase that acts as a regulator of the TORC1 signaling pathway (PubMed:33594058, PubMed:35114100). Positively regulates the TORC1 signaling pathway independently of arginine levels: acts by catalyzing 'Lys-29'-polyubiquitination and degradation of CASTOR1, releasing the GATOR2 complex from CASTOR1 (PubMed:33594058). Also negatively regulates the TORC1 signaling pathway in response to leucine deprivation: acts by mediating 'Lys-63'-linked polyubiquitination of SESN2, promoting SESN2-interaction with the GATOR2 complex (PubMed:35114100). Also involved in protein trafficking and localization (PubMed:23129617, PubMed:23353890, PubMed:24387786, PubMed:27808481, PubMed:32409562). Acts as a regulator of synaptic transmission by mediating ubiquitination and degradation of AMPAR receptor GluA2/GRIA2 (PubMed:23129617, PubMed:33650289). Does not catalyze ubiquitination of GluA1/GRIA1 (PubMed:23129617). Also acts as a regulator of the recycling endosome pathway by mediating ubiquitination of VAMP3 (PubMed:23353890). Regulates lysosome positioning by catalyzing ubiquitination and degradation of ARL8B (PubMed:27808481). Plays a role in growth regulation involved in G1/S transition by mediating, possibly by mediating ubiquitination of SLC22A18 (PubMed:16314844). Acts with a limited set of E2 enzymes, such as UBE2D1 and UBE2N (PubMed:33650289)","subcellular_location":"Cytoplasm, cytosol","url":"https://www.uniprot.org/uniprotkb/Q9H6Y7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RNF167","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"LAMP1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/RNF167","total_profiled":1310},"omim":[{"mim_id":"610431","title":"RING FINGER PROTEIN 167; RNF167","url":"https://www.omim.org/entry/610431"},{"mim_id":"602631","title":"SOLUTE CARRIER FAMILY 67, MEMBER 1; SLC67A1","url":"https://www.omim.org/entry/602631"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Vesicles","reliability":"Approved"},{"location":"Cytosol","reliability":"Approved"},{"location":"Mitotic spindle","reliability":"Additional"},{"location":"Centriolar satellite","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/RNF167"},"hgnc":{"alias_symbol":["DKFZP566H073"],"prev_symbol":[]},"alphafold":{"accession":"Q9H6Y7","domains":[{"cath_id":"3.50.30.30","chopping":"25-167","consensus_level":"high","plddt":93.762,"start":25,"end":167},{"cath_id":"3.30.40.10","chopping":"211-265","consensus_level":"high","plddt":89.0089,"start":211,"end":265}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H6Y7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H6Y7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H6Y7-F1-predicted_aligned_error_v6.png","plddt_mean":78.44},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RNF167","jax_strain_url":"https://www.jax.org/strain/search?query=RNF167"},"sequence":{"accession":"Q9H6Y7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9H6Y7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9H6Y7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H6Y7"}},"corpus_meta":[{"pmid":"35114100","id":"PMC_35114100","title":"E3 ligase RNF167 and deubiquitinase STAMBPL1 modulate mTOR and cancer progression.","date":"2022","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/35114100","citation_count":71,"is_preprint":false},{"pmid":"23129617","id":"PMC_23129617","title":"Ubiquitin ligase RNF167 regulates AMPA receptor-mediated synaptic transmission.","date":"2012","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/23129617","citation_count":69,"is_preprint":false},{"pmid":"33594058","id":"PMC_33594058","title":"RNF167 activates mTORC1 and promotes tumorigenesis by targeting CASTOR1 for ubiquitination and degradation.","date":"2021","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/33594058","citation_count":41,"is_preprint":false},{"pmid":"24387786","id":"PMC_24387786","title":"Tumour-associated mutations of PA-TM-RING ubiquitin ligases RNF167/RNF13 identify the PA domain as a determinant for endosomal localization.","date":"2014","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/24387786","citation_count":30,"is_preprint":false},{"pmid":"27808481","id":"PMC_27808481","title":"RNF167 targets Arl8B for degradation to regulate lysosome positioning and endocytic trafficking.","date":"2016","source":"The FEBS journal","url":"https://pubmed.ncbi.nlm.nih.gov/27808481","citation_count":24,"is_preprint":false},{"pmid":"33650289","id":"PMC_33650289","title":"Functional interaction of ubiquitin ligase RNF167 with UBE2D1 and UBE2N promotes ubiquitination of AMPA receptor.","date":"2021","source":"The FEBS journal","url":"https://pubmed.ncbi.nlm.nih.gov/33650289","citation_count":12,"is_preprint":false},{"pmid":"37410058","id":"PMC_37410058","title":"RNF167-mediated ubiquitination of Tollip inhibits TNF-α-triggered NF-κB and MAPK activation.","date":"2023","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/37410058","citation_count":10,"is_preprint":false},{"pmid":"35887194","id":"PMC_35887194","title":"Membrane Targeting and GTPase Activity of Rab7 Are Required for Its Ubiquitination by RNF167.","date":"2022","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/35887194","citation_count":6,"is_preprint":false},{"pmid":"39994288","id":"PMC_39994288","title":"RNF167 mediates atypical ubiquitylation and degradation of RLRs via two distinct proteolytic pathways.","date":"2025","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/39994288","citation_count":3,"is_preprint":false},{"pmid":"40751214","id":"PMC_40751214","title":"SMAD3 orchestrates RNF167 and STAMBPL1-mediated Sestrin2 ubiquitination to drive gastric cancer progression.","date":"2025","source":"Cell division","url":"https://pubmed.ncbi.nlm.nih.gov/40751214","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.06.20.660705","title":"RNF13 is a novel interactor of iduronate 2-sulfatase that modifies its glycosylation and maturation","date":"2025-06-21","source":"bioRxiv","url":"https://doi.org/10.1101/2025.06.20.660705","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":6683,"output_tokens":3001,"usd":0.032532},"stage2":{"model":"claude-opus-4-6","input_tokens":6348,"output_tokens":2605,"usd":0.145297},"total_usd":0.177829,"stage1_batch_id":"msgbatch_01SUR8NDzVrNmyszWyJeCct7","stage2_batch_id":"msgbatch_01G7Yfutko3cDWzejrwH5AMx","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2012,\n      \"finding\": \"RNF167 is a transmembrane RING domain-containing E3 ubiquitin ligase that is predominantly lysosomal (with a subpopulation on the neuronal surface) and ubiquitinates AMPA receptor subunits in an activity-dependent manner, thereby reducing AMPAR surface expression and synaptic AMPAR currents without affecting NMDAR currents.\",\n      \"method\": \"shRNA knockdown, RING-dead mutant overexpression, surface expression assays, electrophysiology in hippocampal neurons, subcellular localization by fluorescence microscopy\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (genetic KD, dominant-negative mutant, electrophysiology, localization) in a single study\",\n      \"pmids\": [\"23129617\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Point mutations in the RING domain of RNF167 abrogate its E3 ubiquitin ligase activity, and the PA (protease-associated) domain is required for endosomal localization; PA domain mutations found in human tumors retain ligase activity but cause mislocalization, indicating both enzymatic activity and correct localization are needed for substrate targeting.\",\n      \"method\": \"Mutagenesis of RING and PA domains, ubiquitination assays, subcellular localization microscopy\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — in vitro ligase assays combined with mutagenesis and localization studies\",\n      \"pmids\": [\"24387786\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"RNF167 ubiquitinates the small GTPase Arl8B at lysine K141, targeting it for degradation; RNF167 overexpression reduces Arl8B levels and alters lysosome positioning and endocytic trafficking to lysosomes, effects that are counteracted by the ubiquitination-defective Arl8B K141R mutant.\",\n      \"method\": \"Proximity-dependent biotin labeling (BioID) for substrate identification, in vitro ubiquitination assay, site-directed mutagenesis (K141R), overexpression/knockdown with lysosome positioning and endocytic trafficking readouts\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — substrate identified by BioID, confirmed by in vitro ubiquitination and mutagenesis with defined cellular phenotype\",\n      \"pmids\": [\"27808481\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RNF167 ubiquitinates CASTOR1 with K29-linked polyubiquitin chains, leading to its proteasomal degradation; AKT phosphorylates CASTOR1 at S14, which increases CASTOR1 binding to RNF167 and promotes its ubiquitination/degradation while reducing its binding to MIOS, thereby activating mTORC1 in an arginine-independent manner.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitination assays, ubiquitin linkage-specific analysis, phosphorylation assays, mTORC1 activity readouts, knockdown/knockout with xenograft tumor models\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — multiple orthogonal biochemical methods with in vivo functional validation\",\n      \"pmids\": [\"33594058\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RNF167 functionally interacts with E2 conjugating enzymes UBE2D1 and UBE2N; UBE2N drives K63-linked polyubiquitination of GluA2 (AMPAR subunit) only after GluA2 has been primed by a UBE2D1-initiated monoubiquitin, and pharmacological inhibition of UBE2N in hippocampal neurons reduces AMPA-induced GluA2 ubiquitination.\",\n      \"method\": \"In vitro autoubiquitination assays, binding assays with kinetic analysis, fluorescence microscopy co-localization, computational modeling of RING–E2 interaction, pharmacological inhibition of UBE2N in primary neurons\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution of polyubiquitination cascade with mechanistic dissection of E2 contributions, replicated in neurons\",\n      \"pmids\": [\"33650289\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RNF167 (E3) and deubiquitinase STAMBPL1 oppositely control polyubiquitination of Sestrin2; RNF167-mediated ubiquitination of Sestrin2 promotes its interaction with GATOR2 and inhibits mTORC1 signaling in response to leucine availability.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, mTORC1 activity measurements, CRISPR knockout and heterozygous mutation correction, cell-permeable peptide blocking STAMBPL1-Sestrin2 interaction, xenograft tumor models\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — multiple orthogonal biochemical and cellular methods, in vivo tumor models, functional peptide validation\",\n      \"pmids\": [\"35114100\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RNF167 ubiquitinates Rab7 in a manner dependent on Rab7 being in its GTP-bound active (membrane-anchored) form; RNF167-mediated ubiquitination affects Rab7 membrane localization and results in larger Rab7-positive vesicles, and Charcot-Marie-Tooth Type 2B disease variants of Rab7 impair RNF167-mediated ubiquitination.\",\n      \"method\": \"Immunoprecipitation, in vitro ubiquitination assays, subcellular fractionation, epifluorescence microscopy, RNF167 knockdown with Lamp1 and vesicle size readouts\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple methods but single lab, no mutagenesis of ubiquitination sites on Rab7\",\n      \"pmids\": [\"35887194\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"RNF167 interacts with Tollip via Tollip's CUE domain and attaches K33-linked polyubiquitin chains to Tollip at K235; this ubiquitination is required for Tollip to suppress TNF-α-induced NF-κB and MAPK (JNK) signaling, as the K235R Tollip mutant fails to inhibit these cascades.\",\n      \"method\": \"Co-immunoprecipitation, in vitro/cellular ubiquitination assays, ubiquitin linkage analysis, K235R point mutagenesis, NF-κB/MAPK reporter/kinase assays\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP and mutagenesis with defined signaling phenotype, single lab\",\n      \"pmids\": [\"37410058\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RNF167 attaches atypical K6-linked polyubiquitin chains to the CARD domains of RIG-I and MDA5, marking them for p62-mediated selective autophagic degradation in autolysosomes; it also attaches K11-linked polyubiquitin chains to the CTD domains of RIG-I/MDA5, targeting them for proteasomal degradation. Both pathways synergistically suppress RLR-triggered type I interferon signaling.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitination assays, ubiquitin linkage-specific analysis, domain-specific mutagenesis, autophagy flux assays (p62 interaction), proteasome inhibitor experiments, IFN-I reporter assays, RNF167 knockout\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — mechanistically dissects two distinct ubiquitin linkages on two distinct domains with orthogonal degradation pathway readouts\",\n      \"pmids\": [\"39994288\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RNF167 forms a heterodimer with the related E3 ubiquitin ligase RNF13, and this heterodimer modifies both RNF13 and RNF167 lysosomal trafficking; the heterodimer interacts with and alters IDS (iduronate 2-sulfatase) glycosylation and maturation differently than either protein alone, though RNF167's catalytic activity is not required for generating the underglycosylated IDS form.\",\n      \"method\": \"Co-immunoprecipitation, AlphaFold3 structural prediction (functional co-IP validation), catalytic mutant analysis, glycosylation and lysosomal trafficking assays\",\n      \"journal\": \"bioRxiv (preprint)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — preprint, single Co-IP plus mutagenesis, no independent replication\",\n      \"pmids\": [\"bio_10.1101_2025.06.20.660705\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SMAD3 stabilizes Sestrin2 protein by modulating the balance between RNF167-mediated ubiquitination and STAMBPL1-mediated deubiquitination of Sestrin2, as shown by co-immunoprecipitation of SMAD3 with both enzymes.\",\n      \"method\": \"Co-immunoprecipitation, Western blotting, RT-qPCR, immunofluorescence, SMAD3 overexpression/knockdown\",\n      \"journal\": \"Cell division\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP without in vitro reconstitution; regulatory placement is correlative\",\n      \"pmids\": [\"40751214\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RNF167 is a transmembrane RING-type E3 ubiquitin ligase that resides in endosomes and lysosomes (PA domain-dependent localization) and ubiquitinates a range of substrates—including AMPAR subunits (via UBE2D1/UBE2N), Arl8B, Rab7, CASTOR1, Sestrin2, Tollip, and RIG-I/MDA5—using distinct ubiquitin chain linkages (K6, K11, K29, K33, K63) to regulate lysosome positioning, endocytic trafficking, mTORC1 signaling, synaptic strength, NF-κB/MAPK signaling, and antiviral innate immune responses.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"RNF167 is a transmembrane RING-type E3 ubiquitin ligase that resides on endosomes and lysosomes—dependent on its protease-associated (PA) domain—and ubiquitinates diverse substrates using atypical ubiquitin chain linkages to regulate endolysosomal trafficking, mTORC1 nutrient sensing, synaptic plasticity, and innate immune signaling. It ubiquitinates AMPA receptor subunits via a two-step mechanism in which UBE2D1 primes monoubiquitination and UBE2N extends K63-linked chains, reducing synaptic AMPAR surface expression [PMID:23129617, PMID:33650289]. RNF167 targets the lysosome-positioning GTPase Arl8B (K141) and GTP-bound Rab7 for ubiquitin-dependent degradation, controlling lysosome distribution and endocytic flux [PMID:27808481, PMID:35887194], and regulates mTORC1 through opposing ubiquitination events on CASTOR1 (K29-linked chains promoting proteasomal degradation to activate mTORC1) and Sestrin2 (polyubiquitination enhancing GATOR2 binding to inhibit mTORC1) [PMID:33594058, PMID:35114100]. It also attaches K6-linked chains to the CARD domains and K11-linked chains to the CTD domains of RIG-I and MDA5, directing them to autophagic and proteasomal degradation respectively, thereby suppressing type I interferon signaling [PMID:39994288].\",\n  \"teleology\": [\n    {\n      \"year\": 2012,\n      \"claim\": \"Establishing RNF167 as a lysosomal E3 ligase that controls synaptic AMPAR levels answered the question of whether endolysosomal ubiquitin ligases directly regulate ionotropic receptor trafficking at synapses.\",\n      \"evidence\": \"shRNA knockdown, RING-dead mutant, surface expression assays, and electrophysiology in hippocampal neurons\",\n      \"pmids\": [\"23129617\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitination sites on AMPAR subunits not mapped\", \"E2 conjugating enzymes not identified\", \"Mechanism linking lysosomal RNF167 to surface receptor removal unclear\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Demonstrating that the PA domain controls endosomal targeting while the RING domain provides catalytic activity resolved how RNF167 couples its enzymatic function to correct subcellular localization, and showed that cancer-associated PA mutations cause mislocalization without loss of ligase activity.\",\n      \"evidence\": \"Systematic mutagenesis of RING and PA domains with in vitro ubiquitination assays and subcellular localization microscopy\",\n      \"pmids\": [\"24387786\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of PA domain–membrane interaction unknown\", \"Whether PA-mutant mislocalization contributes to tumorigenesis not tested in vivo\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identifying Arl8B as a direct RNF167 substrate ubiquitinated at K141 established a mechanism by which RNF167 controls lysosome positioning and endocytic trafficking to lysosomes.\",\n      \"evidence\": \"BioID proximity labeling for substrate discovery, in vitro ubiquitination, K141R mutagenesis, lysosome positioning and trafficking assays\",\n      \"pmids\": [\"27808481\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitin chain type on Arl8B not determined\", \"Whether Arl8B degradation is proteasomal or lysosomal not resolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Two studies revealed the E2 selectivity of RNF167 (UBE2D1 primes, UBE2N extends K63 chains on GluA2) and identified CASTOR1 as a substrate whose K29-linked ubiquitination by RNF167 integrates AKT phosphorylation with mTORC1 activation, answering how RNF167 builds specific ubiquitin chain types and connects to nutrient signaling.\",\n      \"evidence\": \"In vitro reconstitution of E2–E3 ubiquitination cascade with kinetic analysis and pharmacological UBE2N inhibition in neurons; Co-IP, ubiquitin linkage analysis, phosphorylation assays, and xenograft models for CASTOR1\",\n      \"pmids\": [\"33650289\", \"33594058\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether UBE2D1/UBE2N pairing applies to non-AMPAR substrates unknown\", \"Structural basis of RNF167–CASTOR1 recognition not determined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Discovery that RNF167 ubiquitinates Sestrin2 to promote GATOR2 interaction and suppress mTORC1 in response to leucine, and ubiquitinates GTP-bound Rab7 affecting late endosome morphology, broadened RNF167's role to nutrient-responsive mTORC1 inhibition and endosomal GTPase regulation.\",\n      \"evidence\": \"CRISPR knockout, cell-permeable peptide, xenograft models for Sestrin2; in vitro ubiquitination, subcellular fractionation, vesicle size analysis for Rab7\",\n      \"pmids\": [\"35114100\", \"35887194\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitin chain type on Rab7 not determined\", \"Ubiquitination sites on Rab7 not mapped\", \"How RNF167 exerts opposing effects on mTORC1 via CASTOR1 versus Sestrin2 is not integrated\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identification of Tollip as a substrate receiving K33-linked ubiquitin at K235, required for Tollip-mediated suppression of TNF-α-induced NF-κB and JNK signaling, expanded RNF167's repertoire to inflammatory signaling regulation via atypical chain types.\",\n      \"evidence\": \"Reciprocal Co-IP, ubiquitin linkage analysis, K235R mutagenesis, NF-κB/MAPK reporter assays\",\n      \"pmids\": [\"37410058\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab finding awaiting independent replication\", \"Whether RNF167–Tollip axis operates in vivo during inflammation not tested\", \"Mechanism of K33 chain recognition by downstream effectors unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Mechanistic dissection of dual ubiquitin chain types on RIG-I/MDA5 (K6-linked on CARD for autophagic degradation via p62; K11-linked on CTD for proteasomal degradation) revealed how RNF167 suppresses antiviral innate immunity through two synergistic degradation pathways.\",\n      \"evidence\": \"Domain-specific mutagenesis, linkage-specific ubiquitin analysis, autophagy flux assays, proteasome inhibitor experiments, IFN-I reporters, RNF167 knockout\",\n      \"pmids\": [\"39994288\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance during viral infection not demonstrated\", \"How RNF167 selects K6 versus K11 linkages on different domains mechanistically unclear\", \"E2 enzymes mediating K6 and K11 chain assembly not identified\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include how RNF167 coordinates opposing effects on mTORC1 (CASTOR1 degradation activates, Sestrin2 ubiquitination inhibits), the structural basis of PA domain-mediated membrane targeting, and whether the RNF167–RNF13 heterodimer represents a physiologically relevant complex with distinct substrate specificity.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structural model of full-length RNF167 or its PA domain–membrane interaction\", \"Context-dependent regulation of RNF167 toward opposing mTORC1 outcomes not resolved\", \"RNF167–RNF13 heterodimer function supported only by preprint data\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 2, 3, 4, 5, 6, 7, 8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [1, 6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 5, 7]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 2, 3, 4, 5, 6, 7, 8]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [0, 4]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [2, 6]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"UBE2D1\",\n      \"UBE2N\",\n      \"ARL8B\",\n      \"RAB7A\",\n      \"CASTOR1\",\n      \"SESN2\",\n      \"TOLLIP\",\n      \"DDX58\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}