id: Q96A37 gene_symbol: RNF166 product_type: PROTEIN status: COMPLETE taxon: id: NCBITaxon:9606 label: Homo sapiens description: >- RNF166 (RING finger protein 166) is a small (237 aa) RING-type E3 ubiquitin-protein ligase (EC 2.3.2.27) of the C3HC4 RING subfamily that also includes RNF114, RNF125 and RNF138. It is built from an N-terminal RING-type zinc finger (residues ~33-73; catalytic Cys33/Cys36) that recruits a ubiquitin-charged E2 conjugating enzyme (UBE2D2/UBE2D family), a central C2HC RNF-type zinc finger (~98-117) that mediates substrate/target binding, and a C-terminal ubiquitin-interacting motif (UIM). RNF166 catalyzes ubiquitin transfer to several substrates using different, largely non-degradative chain linkages. In antibacterial selective autophagy (xenophagy) it directly catalyzes Lys29- and Lys33-linked polyubiquitination of the autophagy adaptor SQSTM1/p62 (at K91 and K189), and is required for the early recruitment of ubiquitin and the autophagy adaptors p62 and NDP52 (and downstream LC3) to cytosol-invading bacteria such as Listeria monocytogenes and Shigella flexneri, thereby restricting their intracellular replication. In innate antiviral immunity RNF166 enhances RNA virus-induced Lys63-linked ubiquitination of the signaling adaptors TRAF3 and TRAF6, acting downstream of the mitochondrial adaptor MAVS/VISA and upstream of TBK1 to potentiate type I interferon (IFN-beta) production. RNF166 is a cytoplasmic protein that forms cytosolic puncta and relocalizes to intracellular bacteria. Its catalytic RING activity (lost in the C33A/C36A ligase-dead mutant) is required for both the xenophagy and antiviral functions. alternative_products: - name: '1' id: Q96A37-1 - name: '2' id: Q96A37-2 sequence_note: VSP_019750 - name: '3' id: Q96A37-3 sequence_note: VSP_046147 existing_annotations: - term: id: GO:0000209 label: protein polyubiquitination evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: involved_in review: summary: Phylogenetic inference of protein polyubiquitination, the core catalytic process of RNF166, which assembles polyubiquitin chains on its substrates. action: ACCEPT reason: Core biological process directly demonstrated experimentally - RNF166 catalyzes K29/K33-linked polyubiquitination of p62 and enhances K63-linked polyubiquitination of TRAF3/TRAF6. supported_by: - reference_id: PMID:27880896 supporting_text: RNF166 catalyzes K29- and K33-linked polyubiquitination of p62 at residues K91 and K189 - reference_id: PMID:26456228 supporting_text: K63-linked rather than K48-linked ubiquitination of TRAF3 and TRAF6 was decreased upon SeV infection when RNF166 was knocked down - term: id: GO:0006511 label: ubiquitin-dependent protein catabolic process evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: involved_in review: summary: Phylogenetic (family-level) inference of a degradative ubiquitin-dependent catabolic role. The experimentally established RNF166 ubiquitination events are non-degradative (K29/K33 on p62 promoting xenophagy; K63 on TRAF3/TRAF6 promoting signaling), so a proteasomal/catabolic framing is a family-default that does not match the gene-specific biology. action: MARK_AS_OVER_ANNOTATED reason: RNF166's verified substrates are modified with non-degradative chain types serving autophagy and innate-immune signaling, not proteasomal degradation; the catabolic-process term is an over-propagated IBA default. No experimental evidence shows RNF166 targets a substrate for ubiquitin-dependent degradation. supported_by: - reference_id: PMID:27880896 supporting_text: these data suggest that RNF166 drives K29- and K33-linked ubiquitination of p62 - term: id: GO:0061630 label: ubiquitin protein ligase activity evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: enables review: summary: Phylogenetic inference of ubiquitin protein ligase activity, the core molecular function of RNF166 as a genuine RING-type E3 ligase. action: ACCEPT reason: Core molecular function corroborated by direct in vitro and cell-based evidence; the ligase-dead C33A/C36A RING mutant abolishes activity. supported_by: - reference_id: PMID:27880896 supporting_text: a ligase-dead RNF166 mutant (RNF166 C33A, C36A), was unable to drive p62 ubiquitination under the same conditions - term: id: GO:0005737 label: cytoplasm evidence_type: IEA original_reference_id: GO_REF:0000044 qualifier: located_in review: summary: Electronic transfer of cytoplasmic localization from the UniProt subcellular location; the active compartment of RNF166, corroborated by experimental imaging. action: ACCEPT reason: Correct core localization; RNF166 is cytoplasmic, forming cytosolic puncta and relocalizing to intracellular bacteria. Redundant with the EXP cytoplasm annotation. supported_by: - reference_id: PMID:26456228 supporting_text: Overexpressed RNF166 localized predominantly in the cytosol as dots which were probably aggregates of RNF166 protein - term: id: GO:0061630 label: ubiquitin protein ligase activity evidence_type: IEA original_reference_id: GO_REF:0000003 qualifier: enables review: summary: Enzyme Commission-based electronic assignment of ubiquitin protein ligase activity (EC 2.3.2.27), the core catalytic molecular function. action: ACCEPT reason: Core molecular function; EC 2.3.2.27 was experimentally established for RNF166. Redundant with the IBA/EXP ligase activity annotations. supported_by: - reference_id: file:human/RNF166/RNF166-uniprot.txt supporting_text: EC=2.3.2.27 {ECO:0000269|PubMed:27880896} - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:19549727 qualifier: enables review: summary: Interaction from the human E2 ubiquitin-conjugating enzyme interaction network (E2 binding). Bare protein binding is uninformative. action: KEEP_AS_NON_CORE reason: Records a real interaction (consistent with RNF166 binding E2 enzymes such as UBE2D4/UBE2K, central to its RING mechanism) but bare protein binding is uninformative per curation guidelines. supported_by: - reference_id: file:human/RNF166/RNF166-uniprot.txt supporting_text: 'Q96A37; Q9Y2X8: UBE2D4; NbExp=4; IntAct=EBI-2130320, EBI-745527' - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:32296183 qualifier: enables review: summary: High-throughput binary interactome interaction. Bare protein binding is uninformative. action: KEEP_AS_NON_CORE reason: High-throughput interactome; bare protein binding is uninformative per curation guidelines. supported_by: - reference_id: file:human/RNF166/RNF166-uniprot.txt supporting_text: 'SUBCELLULAR LOCATION: Cytoplasm {ECO:0000269|PubMed:26456228}' - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:32814053 qualifier: enables review: summary: Neurodegeneration interactome interaction (TARDBP). Bare protein binding is uninformative. action: KEEP_AS_NON_CORE reason: Records a real interaction (TARDBP) but bare protein binding is uninformative per curation guidelines. supported_by: - reference_id: file:human/RNF166/RNF166-uniprot.txt supporting_text: 'Q96A37; Q13148: TARDBP; NbExp=3; IntAct=EBI-2130320, EBI-372899' - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:33961781 qualifier: enables review: summary: Cell-specific interactome interaction. Bare protein binding is uninformative. action: KEEP_AS_NON_CORE reason: High-throughput interactome; bare protein binding is uninformative per curation guidelines. supported_by: - reference_id: file:human/RNF166/RNF166-uniprot.txt supporting_text: 'SUBCELLULAR LOCATION: Cytoplasm {ECO:0000269|PubMed:26456228}' - term: id: GO:0016567 label: protein ubiquitination evidence_type: IEA original_reference_id: GO_REF:0000041 qualifier: involved_in review: summary: UniPathway-derived general protein ubiquitination process, a parent of the specific polyubiquitination RNF166 catalyzes. action: KEEP_AS_NON_CORE reason: Correct but generic; the specific protein polyubiquitination annotation (GO:0000209) better captures the role. supported_by: - reference_id: file:human/RNF166/RNF166-uniprot.txt supporting_text: 'PATHWAY: Protein modification; protein ubiquitination.' - term: id: GO:0005737 label: cytoplasm evidence_type: EXP original_reference_id: PMID:26456228 qualifier: located_in review: summary: Experimental evidence (immunofluorescence) that RNF166 localizes to the cytoplasm, forming cytosolic dots. The active compartment for its xenophagy and antiviral functions. action: ACCEPT reason: Core localization with direct experimental support; RNF166 acts in the cytoplasm where it ubiquitinates p62 on bacteria and TRAF3/TRAF6 in antiviral signaling. supported_by: - reference_id: PMID:26456228 supporting_text: Overexpressed RNF166 localized predominantly in the cytosol as dots which were probably aggregates of RNF166 protein - term: id: GO:0061630 label: ubiquitin protein ligase activity evidence_type: EXP original_reference_id: PMID:27880896 qualifier: enables review: summary: Experimental evidence that RNF166 is a ubiquitin protein ligase - reconstituted in vitro with E1, UBE2D2 (E2) and ubiquitin to ubiquitinate p62, with activity abolished in the RING ligase-dead mutant. Core molecular function. action: ACCEPT reason: Core molecular function directly demonstrated; RNF166 is a genuine catalytic RING-type E3 ligase whose activity requires an intact RING (C33/C36). supported_by: - reference_id: PMID:27880896 supporting_text: we used an in vitro ubiquitination assay with recombinant UBA1 (E1), E2 enzymes, HA-ubiquitin, GST-RNF166, and SUMO-p62 references: - id: GO_REF:0000003 title: Gene Ontology annotation based on Enzyme Commission mapping findings: [] - id: GO_REF:0000033 title: Annotation inferences using phylogenetic trees findings: [] - id: GO_REF:0000041 title: Gene Ontology annotation based on UniPathway vocabulary mapping findings: [] - id: GO_REF:0000044 title: Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping, accompanied by conservative changes to GO terms applied by UniProt findings: [] - id: PMID:19549727 title: Analysis of the human E2 ubiquitin conjugating enzyme protein interaction network. findings: [] reference_review: relevance: LOW correctness: VERIFIED review_notes: High-throughput E2 interaction network; source of a bare protein binding annotation, consistent with RNF166 binding E2 conjugating enzymes. - id: PMID:26456228 title: Ring finger protein 166 potentiates RNA virus-induced interferon-β production via enhancing the ubiquitination of TRAF3 and TRAF6. findings: - statement: RNF166 potentiates RNA virus-induced IFN-beta production by enhancing K63-linked ubiquitination of TRAF3 and TRAF6, acting downstream of MAVS/VISA and upstream of TBK1; RNF166 is cytoplasmic and its RING domain is required. reference_section_type: RESULTS reference_review: relevance: HIGH correctness: VERIFIED review_notes: Full text available; establishes the innate antiviral (TRAF3/TRAF6, type I IFN) role and cytoplasmic localization. - id: PMID:27880896 title: RNF166 Determines Recruitment of Adaptor Proteins during Antibacterial Autophagy. findings: - statement: RNF166 is a catalytic RING E3 ligase that directly ubiquitinates SQSTM1/p62 via K29/K33-linked chains (at K91/K189), is required for early recruitment of ubiquitin, p62 and NDP52 to cytosolic bacteria, and restricts intracellular replication of Listeria and Shigella; the RING ligase-dead mutant (C33A/C36A) abolishes p62 ubiquitination. reference_section_type: RESULTS reference_review: relevance: HIGH correctness: VERIFIED review_notes: Full text available; the definitive functional/mechanistic study establishing RNF166's catalytic activity and xenophagy role. - id: PMID:32296183 title: A reference map of the human binary protein interactome. findings: [] reference_review: relevance: LOW correctness: VERIFIED review_notes: Binary interactome reference map; source of a bare protein binding annotation. - id: PMID:32814053 title: Interactome Mapping Provides a Network of Neurodegenerative Disease Proteins and Uncovers Widespread Protein Aggregation in Affected Brains. findings: [] reference_review: relevance: LOW correctness: VERIFIED review_notes: Neurodegeneration interactome; source of a bare protein binding (TARDBP) annotation. - id: PMID:33961781 title: Dual proteome-scale networks reveal cell-specific remodeling of the human interactome. findings: [] reference_review: relevance: LOW correctness: VERIFIED review_notes: Cell-specific interactome; source of a bare protein binding annotation. core_functions: - description: Functions as a catalytic RING-type E3 ubiquitin-protein ligase that recruits a ubiquitin-charged E2 (UBE2D family) and transfers ubiquitin to substrate lysines, assembling polyubiquitin chains. Catalytic activity requires the intact RING (Cys33/Cys36). molecular_function: id: GO:0061630 label: ubiquitin protein ligase activity locations: - id: GO:0005737 label: cytoplasm supported_by: - reference_id: PMID:27880896 supporting_text: a ligase-dead RNF166 mutant (RNF166 C33A, C36A), was unable to drive p62 ubiquitination under the same conditions directly_involved_in: - id: GO:0000209 label: protein polyubiquitination - description: In antibacterial selective autophagy (xenophagy), directly catalyzes K29/K33-linked polyubiquitination of the autophagy adaptor SQSTM1/p62 and drives early recruitment of ubiquitin, p62 and NDP52 to cytosol-adapted bacteria, restricting intracellular replication of Listeria and Shigella. molecular_function: id: GO:0061630 label: ubiquitin protein ligase activity locations: - id: GO:0005737 label: cytoplasm supported_by: - reference_id: PMID:27880896 supporting_text: RNF166 catalyzes K29- and K33-linked polyubiquitination of p62 at residues K91 and K189 directly_involved_in: - id: GO:0000209 label: protein polyubiquitination - description: In innate antiviral immunity, enhances RNA virus-induced K63-linked ubiquitination of the signaling adaptors TRAF3 and TRAF6, acting downstream of MAVS/VISA and upstream of TBK1 to potentiate type I interferon (IFN-beta) production. molecular_function: id: GO:0061630 label: ubiquitin protein ligase activity locations: - id: GO:0005737 label: cytoplasm supported_by: - reference_id: PMID:26456228 supporting_text: RNF166 positively regulates RNA virus-triggered IFN-β production by enhancing the ubiquitination of TRAF3 and TRAF6 directly_involved_in: - id: GO:0000209 label: protein polyubiquitination proposed_new_terms: - proposed_name: xenophagy proposed_definition: The selective degradation of intracellular pathogen or some part of an intracellular pathogen by macroautophagy. justification: RNF166 is directly required for xenophagy of cytosol-adapted bacteria (Listeria, Shigella) - it ubiquitinates p62, recruits autophagy adaptors to bacteria, and restricts their replication - yet only generic catabolic/ubiquitination terms are present in the current GOA. The verified term GO:0098792 (xenophagy) should be annotated. proposed_parent: id: GO:0098792 label: xenophagy supported_by: - reference_id: PMID:27880896 supporting_text: RNF166 functions as an important component of autophagic targeting of cytosol-adapted pathogenic bacteria - proposed_name: defense response to bacterium proposed_definition: Reactions triggered in response to the presence of a bacterium that act to protect the cell or organism. justification: RNF166 restricts intracellular replication of Listeria and Shigella through ligase-dependent xenophagy, a cell-autonomous antibacterial defense not captured by current GOA terms. GO:0042742 should be annotated. proposed_parent: id: GO:0042742 label: defense response to bacterium supported_by: - reference_id: PMID:27880896 supporting_text: RNF166 limits intracellular replication of Shigella - proposed_name: positive regulation of type I interferon production proposed_definition: Any process that activates or increases the frequency, rate, or extent of type I interferon production. justification: RNF166 potentiates RNA virus-induced IFN-beta production by enhancing K63-linked ubiquitination of TRAF3/TRAF6 downstream of MAVS; this innate antiviral signaling role is supported experimentally but absent from the curated GOA. GO:0032481 should be annotated. proposed_parent: id: GO:0032481 label: positive regulation of type I interferon production supported_by: - reference_id: PMID:26456228 supporting_text: RNF166 positively regulates RNA virus-triggered IFN-β production by enhancing the ubiquitination of TRAF3 and TRAF6 suggested_questions: - question: Does RNF166 act as a stand-alone monomeric RING E3 or does it require partners/cofactors for substrate selection between p62 (xenophagy) and TRAF3/TRAF6 (antiviral signaling)? - question: What determines the atypical chain-linkage specificity of RNF166 (K29/K33 on p62 versus K63 on TRAF3/TRAF6), and is this dictated by the E2 used or by substrate context? suggested_experiments: - description: Reconstitute RNF166-mediated ubiquitination in vitro with a panel of E2 enzymes and chain-linkage-specific ubiquitin mutants to define the intrinsic chain specificity for p62 versus TRAF3/TRAF6, comparing wild-type and C33A/C36A RING-dead RNF166. - description: Use RNF166-knockout cells reconstituted with wild-type or ligase-dead RNF166 to test antibacterial (Listeria/Shigella) and antiviral (SeV/EMCV type I IFN) phenotypes in parallel, distinguishing catalytic from scaffolding contributions.