RNF166

UniProt ID: Q96A37
Organism: Homo sapiens
Review Status: COMPLETE
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Gene 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.

Proposed New Ontology Terms

xenophagy

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.

Parent term: xenophagy

Supporting Evidence:

defense response to bacterium

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.

Parent term: defense response to bacterium

Supporting Evidence:

positive regulation of type I interferon production

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.

Parent term: positive regulation of type I interferon production

Supporting Evidence:

Existing Annotations Review

GO Term Evidence Action Reason
GO:0000209 protein polyubiquitination
IBA
GO_REF:0000033 		ACCEPT
Summary: Phylogenetic inference of protein polyubiquitination, the core catalytic process of RNF166, which assembles polyubiquitin chains on its substrates.
Reason: Core biological process directly demonstrated experimentally - RNF166 catalyzes K29/K33-linked polyubiquitination of p62 and enhances K63-linked polyubiquitination of TRAF3/TRAF6.
Supporting Evidence:
PMID:27880896
RNF166 catalyzes K29- and K33-linked polyubiquitination of p62 at residues K91 and K189
PMID:26456228
K63-linked rather than K48-linked ubiquitination of TRAF3 and TRAF6 was decreased upon SeV infection when RNF166 was knocked down
GO:0006511 ubiquitin-dependent protein catabolic process
IBA
GO_REF:0000033 		MARK AS OVER ANNOTATED
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.
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.
Supporting Evidence:
PMID:27880896
these data suggest that RNF166 drives K29- and K33-linked ubiquitination of p62
GO:0061630 ubiquitin protein ligase activity
IBA
GO_REF:0000033 		ACCEPT
Summary: Phylogenetic inference of ubiquitin protein ligase activity, the core molecular function of RNF166 as a genuine RING-type E3 ligase.
Reason: Core molecular function corroborated by direct in vitro and cell-based evidence; the ligase-dead C33A/C36A RING mutant abolishes activity.
Supporting Evidence:
PMID:27880896
a ligase-dead RNF166 mutant (RNF166 C33A, C36A), was unable to drive p62 ubiquitination under the same conditions
GO:0005737 cytoplasm
IEA
GO_REF:0000044 		ACCEPT
Summary: Electronic transfer of cytoplasmic localization from the UniProt subcellular location; the active compartment of RNF166, corroborated by experimental imaging.
Reason: Correct core localization; RNF166 is cytoplasmic, forming cytosolic puncta and relocalizing to intracellular bacteria. Redundant with the EXP cytoplasm annotation.
Supporting Evidence:
PMID:26456228
Overexpressed RNF166 localized predominantly in the cytosol as dots which were probably aggregates of RNF166 protein
GO:0061630 ubiquitin protein ligase activity
IEA
GO_REF:0000003 		ACCEPT
Summary: Enzyme Commission-based electronic assignment of ubiquitin protein ligase activity (EC 2.3.2.27), the core catalytic molecular function.
Reason: Core molecular function; EC 2.3.2.27 was experimentally established for RNF166. Redundant with the IBA/EXP ligase activity annotations.
Supporting Evidence:
file:human/RNF166/RNF166-uniprot.txt
EC=2.3.2.27 {ECO:0000269|PubMed:27880896}
GO:0005515 protein binding
IPI
PMID:19549727
Analysis of the human E2 ubiquitin conjugating enzyme protei... 		KEEP AS NON CORE
Summary: Interaction from the human E2 ubiquitin-conjugating enzyme interaction network (E2 binding). Bare protein binding is uninformative.
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.
Supporting Evidence:
file:human/RNF166/RNF166-uniprot.txt
Q96A37; Q9Y2X8: UBE2D4; NbExp=4; IntAct=EBI-2130320, EBI-745527
GO:0005515 protein binding
IPI
PMID:32296183
A reference map of the human binary protein interactome. 		KEEP AS NON CORE
Summary: High-throughput binary interactome interaction. Bare protein binding is uninformative.
Reason: High-throughput interactome; bare protein binding is uninformative per curation guidelines.
Supporting Evidence:
file:human/RNF166/RNF166-uniprot.txt
SUBCELLULAR LOCATION: Cytoplasm {ECO:0000269|PubMed:26456228}
GO:0005515 protein binding
IPI
PMID:32814053
Interactome Mapping Provides a Network of Neurodegenerative ... 		KEEP AS NON CORE
Summary: Neurodegeneration interactome interaction (TARDBP). Bare protein binding is uninformative.
Reason: Records a real interaction (TARDBP) but bare protein binding is uninformative per curation guidelines.
Supporting Evidence:
file:human/RNF166/RNF166-uniprot.txt
Q96A37; Q13148: TARDBP; NbExp=3; IntAct=EBI-2130320, EBI-372899
GO:0005515 protein binding
IPI
PMID:33961781
Dual proteome-scale networks reveal cell-specific remodeling... 		KEEP AS NON CORE
Summary: Cell-specific interactome interaction. Bare protein binding is uninformative.
Reason: High-throughput interactome; bare protein binding is uninformative per curation guidelines.
Supporting Evidence:
file:human/RNF166/RNF166-uniprot.txt
SUBCELLULAR LOCATION: Cytoplasm {ECO:0000269|PubMed:26456228}
GO:0016567 protein ubiquitination
IEA
GO_REF:0000041 		KEEP AS NON CORE
Summary: UniPathway-derived general protein ubiquitination process, a parent of the specific polyubiquitination RNF166 catalyzes.
Reason: Correct but generic; the specific protein polyubiquitination annotation (GO:0000209) better captures the role.
Supporting Evidence:
file:human/RNF166/RNF166-uniprot.txt
PATHWAY: Protein modification; protein ubiquitination.
GO:0005737 cytoplasm
EXP
PMID:26456228
Ring finger protein 166 potentiates RNA virus-induced interf... 		ACCEPT
Summary: Experimental evidence (immunofluorescence) that RNF166 localizes to the cytoplasm, forming cytosolic dots. The active compartment for its xenophagy and antiviral functions.
Reason: Core localization with direct experimental support; RNF166 acts in the cytoplasm where it ubiquitinates p62 on bacteria and TRAF3/TRAF6 in antiviral signaling.
Supporting Evidence:
PMID:26456228
Overexpressed RNF166 localized predominantly in the cytosol as dots which were probably aggregates of RNF166 protein
GO:0061630 ubiquitin protein ligase activity
EXP
PMID:27880896
RNF166 Determines Recruitment of Adaptor Proteins during Ant... 		ACCEPT
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.
Reason: Core molecular function directly demonstrated; RNF166 is a genuine catalytic RING-type E3 ligase whose activity requires an intact RING (C33/C36).
Supporting Evidence:
PMID:27880896
we used an in vitro ubiquitination assay with recombinant UBA1 (E1), E2 enzymes, HA-ubiquitin, GST-RNF166, and SUMO-p62

Core Functions

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:
ubiquitin protein ligase activity
Directly Involved In:
protein polyubiquitination
Cellular Locations:
cytoplasm
Supporting Evidence:
  • PMID:27880896
    a ligase-dead RNF166 mutant (RNF166 C33A, C36A), was unable to drive p62 ubiquitination under the same conditions

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:
ubiquitin protein ligase activity
Directly Involved In:
protein polyubiquitination
Cellular Locations:
cytoplasm
Supporting Evidence:
  • PMID:27880896
    RNF166 catalyzes K29- and K33-linked polyubiquitination of p62 at residues K91 and K189

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:
ubiquitin protein ligase activity
Directly Involved In:
protein polyubiquitination
Cellular Locations:
cytoplasm
Supporting Evidence:
  • PMID:26456228
    RNF166 positively regulates RNA virus-triggered IFN-Ξ² production by enhancing the ubiquitination of TRAF3 and TRAF6

References

GO_REF:0000003
Gene Ontology annotation based on Enzyme Commission mapping
GO_REF:0000033
Annotation inferences using phylogenetic trees
GO_REF:0000041
Gene Ontology annotation based on UniPathway vocabulary mapping
GO_REF:0000044
Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping, accompanied by conservative changes to GO terms applied by UniProt
PMID:19549727
Analysis of the human E2 ubiquitin conjugating enzyme protein interaction network.
PMID:26456228
Ring finger protein 166 potentiates RNA virus-induced interferon-Ξ² production via enhancing the ubiquitination of TRAF3 and TRAF6.
  • 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.
PMID:27880896
RNF166 Determines Recruitment of Adaptor Proteins during Antibacterial Autophagy.
  • 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.
PMID:32296183
A reference map of the human binary protein interactome.
PMID:32814053
Interactome Mapping Provides a Network of Neurodegenerative Disease Proteins and Uncovers Widespread Protein Aggregation in Affected Brains.
PMID:33961781
Dual proteome-scale networks reveal cell-specific remodeling of the human interactome.

Suggested Questions for Experts

Q: 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)?

Q: 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

Experiment: 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.

Experiment: 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.

πŸ“š Additional Documentation

Notes

(RNF166-notes.md)

RNF166 (RING finger protein 166) review notes

UniProt: Q96A37 (RN166_HUMAN), 237 aa. EC 2.3.2.27. RING-type E3 ubiquitin ligase.
Member of a subfamily of small C3HC4 RING ligases together with RNF114, RNF125, RNF138
PMID:26456228.

Domain architecture

  • RING-type zinc finger 33..73 (catalytic; recruits ubiquitin-charged E2) [UniProt FT].
  • C2HC RNF-type zinc finger 98..117 (mediates substrate/target interaction) [UniProt FT;
    PMID:26456228 "RNF166 interacts with TRAF3 and TRAF6 via its zinc finger domain"].
  • C-terminal UIM (ubiquitin-interacting motif) ~221..237 [UniProt FT].
  • Catalytic Cys33/Cys36 are essential: C33A/C36A is ligase-dead.
    [UniProt MUTAGEN 33/36; PMID:27880896 "a ligase-dead RNF166 mutant (RNF166 C33A, C36A)
    was unable to drive p62 ubiquitination"]

Core molecular function

  • RING-type E3 ubiquitin-protein ligase / ubiquitin-protein transferase (EC 2.3.2.27).
    Demonstrated in vitro with UBA1 (E1), UBE2D2 (E2), HA-ubiquitin, GST-RNF166, SUMO-p62.
    PMID:27880896
  • Zinc ion binding (RING + C2HC Zn fingers) [UniProt KW Zinc-finger; BINDING residues].

Substrates / processes

  • SQSTM1/p62 β€” RNF166 directly catalyzes K29- and K33-linked polyubiquitination of p62 at
    K91 and K189. This is an ATYPICAL (non-degradative) ubiquitination that promotes xenophagy
    (antibacterial selective autophagy) of cytosol-adapted bacteria (Listeria, Shigella).
    [PMID:27880896 "RNF166 catalyzes K29- and K33-linked polyubiquitination of p62 at residues
    K91 and K189"; "RNF166 limits intracellular replication of Shigella and...the E3 ligase
    activity of RNF166 is important for this function"]
  • RNF166 is required for early recruitment of autophagy adaptors p62 and NDP52, and of
    ubiquitin and LC3, to bacteria; localizes to bacteria with p62.
    PMID:27880896
  • NOTE: K29/K33-linked polyubiquitination of p62 here is NOT proteasome-targeting; the
    IBA "ubiquitin-dependent protein catabolic process" / "proteasome" framing from
    GO_Central is the family-level default and only partially fits RNF166's verified biology
    (xenophagy/autophagy, not proteasomal degradation of p62).
  • TRAF3 and TRAF6 β€” RNF166 enhances RNA virus (SeV/EMCV)-induced K63-linked ubiquitination
    of TRAF3 and TRAF6, potentiating VISA(MAVS)-mediated IFN-beta production (innate antiviral
    immunity). RNF166 binds TRAF3/TRAF6 via its zinc finger; RING domain required for the
    positive effect. RNF166 acts downstream of VISA, upstream of TBK1.
    [PMID:26456228 "RNF166...potentiates RNA virus-triggered IFN-beta production by enhancing
    the ubiquitination of TRAF3 and TRAF6"; "K63-linked rather than K48-linked ubiquitination
    of TRAF3 and TRAF6 was decreased...when RNF166 was knocked down"]

Subcellular location

  • Cytoplasm; forms cytosolic dots/aggregates; relocalizes to intracellular bacteria.
    [UniProt SUBCELLULAR LOCATION: Cytoplasm {ECO:0000269|PubMed:26456228}; PMID:26456228
    "Overexpressed RNF166 localized predominantly in the cytosol as dots"]

Interactors (UniProt IntAct)

  • PRKN, TARDBP, UBE2D4, UBE2K, XAF1. UBE2D4/UBE2K are E2 enzymes (consistent with RING E3).

Annotation-call reasoning

  • ubiquitin protein ligase activity (GO:0061630) β€” ACCEPT core MF (EXP/IDA in vitro,
    ligase-dead mutant; IBA also). genuine RING E3.
  • ubiquitin-protein transferase activity (GO:0004842) IEA EC mapping β€” ACCEPT (synonymous
    catalytic MF; EC 2.3.2.27 experimentally established by PMID:27880896).
  • protein polyubiquitination (GO:0000209) IBA β€” ACCEPT core BP (K29/K33 chains on p62; K63
    on TRAF3/6).
  • ubiquitin-dependent protein catabolic process (GO:0006511) IBA β€” MARK_AS_OVER_ANNOTATED:
    RNF166's verified ubiquitination of p62 (K29/K33) and TRAF3/6 (K63) is non-degradative
    (signaling/autophagy), so a generic catabolic/proteasomal framing is a family-level
    over-propagation not supported by the gene-specific evidence. Defer/keep cautious.
  • protein ubiquitination (GO:0016567) IEA UniPathway β€” KEEP_AS_NON_CORE (generic parent of
    the specific polyubiquitination).
  • zinc ion binding (GO:0008270) IEA β€” ACCEPT (RING + C2HC Zn fingers; structurally required).
  • cytoplasm (GO:0005737) IEA + EXP β€” ACCEPT (active compartment; EXP from PMID:26456228).
  • protein binding (GO:0005515) bare IPI β€” KEEP_AS_NON_CORE per curation guidelines.
  • Missing but supportable (not in GOA): autophagy / xenophagy, innate immune response,
    defense response to bacterium, positive regulation of type I IFN production.

Pn Notes

(RNF166-pn-notes.md)

RNF166 PN Consistency Notes

  • Generated: 2026-06-18
  • Project: PROTEOSTASIS
  • Scope: PN consistency rereview against local AIGR review and available deep-research artifacts
  • UniProt: Q96A37
  • AIGR review status: COMPLETE
  • Review batch: proteostasis-batch-2026-06-14
  • Batch change status: added

Source Files Checked

Deep Research Files

  • No *-deep-research*.md file found in this gene directory.

AIGR Review Snapshot

  • 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.
  • Existing/core annotation action counts: ACCEPT: 6; KEEP_AS_NON_CORE: 5; MARK_AS_OVER_ANNOTATED: 1

PN Consistency Summary

  • Consistency: Excellent. Deep research, review and PN all agree RNF166 is a genuine catalytic RING E3 (ligase-dead C33A/C36A abolishes activity) that drives xenophagy of cytosolic bacteria by K29/K33-ubiquitinating p62 and recruiting p62/NDP52 (PMID:27880896), and potentiates antiviral type-I-IFN via K63-Ub of TRAF3/TRAF6 (PMID:26456228). PN row1 leaf ("Catalyzes K33-linked ubiquitination of p62") matches the review exactly. Row3's UIM note matches the UniProt C-terminal UIM. No contradictions.
  • PN story / NEW pressure: Row1 projects GO:0098792 xenophagy (verified real; ABSENT from GOA). This is a strongly defensible ADD β€” and the review itself proposes GO:0098792 as a NEW term (plus GO:0042742 defense response to bacterium and GO:0032481 positive regulation of type I IFN production), fully concordant with the dossier goa_status=new_to_goa. Rows2/3 GO:0061630 = already_in_goa_exact (IBA/EXP/IEA present) β€” keep catalytic ligase MF as core (matches KEY PATTERN). So PN and review agree: ADD xenophagy; ligase MF already captured.
  • Evidence alignment: Strong. PN row1 cites "RNF166 Determines Recruitment of Adaptor Proteins during Antibacterial Autophagy" (= PMID:27880896, the review's HIGH/VERIFIED anchor) plus a p62-UBA-Ub review (tandfonline). Row3 cites PMID:17990982 (not in the review). Review additionally anchors the antiviral arm on PMID:26456228 (not in PN). Minor divergence on antiviral evidence only.
  • Verdict: Fully consistent; xenophagy ADD is justified and the review ALREADY proposes it; catalytic ligase MF already core/in-GOA.

Full Consistency Review

  • UniProt: Q96A37 Β· batch: proteostasis-batch-2026-06-14 Β· review status: COMPLETE (thorough; catalytic C3HC4 RING E3; p62 K29/K33 xenophagy + TRAF3/6 K63 antiviral; 3 PN rows)
  • PN placement: row1 ALP|...|Marking substrates for selective autophagy|Xenophagy|Catalyzes K33-linked ubiquitination of p62; row2 UPS|E3 ligases|RING|TRAC-1; row3 UPS|Ubiquitin and UBL binding|E3 ligase|RING / TRAC-1|UIM. PN-node mapping: row1 Xenophagy typeβ†’mapped GO:0098792 xenophagy; rows2/3 E3-groupβ†’mapped GO:0061630 ubiquitin protein ligase activity (classes context_only/too_broad; subtypes no_mapping).
  • Consistency: Excellent. Deep research, review and PN all agree RNF166 is a genuine catalytic RING E3 (ligase-dead C33A/C36A abolishes activity) that drives xenophagy of cytosolic bacteria by K29/K33-ubiquitinating p62 and recruiting p62/NDP52 (PMID:27880896), and potentiates antiviral type-I-IFN via K63-Ub of TRAF3/TRAF6 (PMID:26456228). PN row1 leaf ("Catalyzes K33-linked ubiquitination of p62") matches the review exactly. Row3's UIM note matches the UniProt C-terminal UIM. No contradictions.
  • PN story / NEW pressure: Row1 projects GO:0098792 xenophagy (verified real; ABSENT from GOA). This is a strongly defensible ADD β€” and the review itself proposes GO:0098792 as a NEW term (plus GO:0042742 defense response to bacterium and GO:0032481 positive regulation of type I IFN production), fully concordant with the dossier goa_status=new_to_goa. Rows2/3 GO:0061630 = already_in_goa_exact (IBA/EXP/IEA present) β€” keep catalytic ligase MF as core (matches KEY PATTERN). So PN and review agree: ADD xenophagy; ligase MF already captured.
  • Mapping strategy: Correct. Catalytic RINGβ†’GO:0061630 (exact, in GOA); Xenophagy typeβ†’GO:0098792 (narrower-but-inside). Subtype p62-K33 leaf and the TRAC-1/UIM architecture subdivisions appropriately no_mapping. The two separate RING/E3 rows both projecting GO:0061630 are redundant but harmless (one MF). No over-reach.
  • Evidence alignment: Strong. PN row1 cites "RNF166 Determines Recruitment of Adaptor Proteins during Antibacterial Autophagy" (= PMID:27880896, the review's HIGH/VERIFIED anchor) plus a p62-UBA-Ub review (tandfonline). Row3 cites PMID:17990982 (not in the review). Review additionally anchors the antiviral arm on PMID:26456228 (not in PN). Minor divergence on antiviral evidence only.
  • Verdict: Fully consistent; xenophagy ADD is justified and the review ALREADY proposes it; catalytic ligase MF already core/in-GOA.
  • Recommended edits: [YAML] none required for the PN story β€” the review already proposes GO:0098792 xenophagy (NEW). Optionally [REF] check PN row3's PMID:17990982 (UIM characterization), absent from the review, if a UIM ubiquitin-reader MF is desired.

PN Dossier Context

  • review_batch: proteostasis-batch-2026-06-14
  • review_yaml: genes/human/RNF166/RNF166-ai-review.yaml
  • PN workbook rows: 3

PN row 1: Autophagy-Lysosome Pathway | Autophagy substrate selection | Marking substrates for selective autophagy | Xenophagy | Catalyzes K33-linked ubiquitination of p62

  • UniProt: Q96A37
  • In branches: ALP, UPS
  • Notes: E3 ubuquitin ligases that ubiquitinate bacteria to enable the recruitment of bacteria cargo by SQSTM1 and CALCOCO2 to ubiquitinated bacteria.
  • PN references (titles):
    • Regulation of SQSTM1/p62 via UBA domain ubiquitination and its role in disease (tandfonline.com)
    • RNF166 Determines Recruitment of Adaptor Proteins during Antibacterial Autophagy
  • PN-node mapping records (path + ancestors):
    • [subtype] Autophagy-Lysosome Pathway|Autophagy substrate selection|Marking substrates for selective autophagy|Xenophagy|Catalyzes K33-linked ubiquitination of p62
      status=no_mapping scope= GO=[]
      rationale: Reviewed as a contextual PN role. The label is useful for curator triage, but by itself does not support a universal GO assertion for all member genes beyond curated ancestor or child mappings.
    • [type] Autophagy-Lysosome Pathway|Autophagy substrate selection|Marking substrates for selective autophagy|Xenophagy
      status=mapped scope=ok_for_propagation_to_go GO=[GO:0098792 xenophagy]
      rationale: This PN type groups xenophagy-specific marking steps that label cargo for selective autophagic clearance. That is narrower than, but clearly inside, the xenophagy process.
    • [group] Autophagy-Lysosome Pathway|Autophagy substrate selection|Marking substrates for selective autophagy
      status=no_mapping scope= GO=[]
      rationale: Reviewed as a broad PN taxonomy container. The descendants mix components, regulators, context labels, and mechanistic leaves, so propagation should come only from narrower curated nodes.
    • [class] Autophagy-Lysosome Pathway|Autophagy substrate selection
      status=no_mapping scope= GO=[]
      rationale: Reviewed as a broad substrate-selection container. GO has useful targets for specific receptor, cargo-adaptor, and selective-autophagy leaves, but this class mixes marking, recognition, receptor regulation, and unknown roles and should not propagate as one term.
    • [branch] Autophagy-Lysosome Pathway
      status=no_mapping scope= GO=[]
      rationale: Reviewed as the top-level PN branch. It is a project taxonomy umbrella rather than a direct GO assertion; all propagation must come from manually curated child nodes.

PN row 2: Ubiquitin Proteasome System | E3 ubiquitin and UBL ligases | RING | TRAC-1

  • UniProt: Q96A37
  • In branches: ALP, UPS
  • Signature domains: IPR001841
  • Auxiliary domains: IPR008598, IPR034734
  • PN references (titles):
    • 19489725 / rev
  • PN-node mapping records (path + ancestors):
    • [type] Ubiquitin Proteasome System|E3 ubiquitin and UBL ligases|RING|TRAC-1
      status=no_mapping scope= GO=[]
      rationale: Reviewed as a narrower E3-ligase architecture, component, or domain subdivision already covered by the curated parent E3 mapping. No additional direct GO mapping is needed at this node.
    • [group] Ubiquitin Proteasome System|E3 ubiquitin and UBL ligases|RING
      status=mapped scope=ok_for_propagation_to_go GO=[GO:0061630 ubiquitin protein ligase activity]
      rationale: This PN group is a catalytic ubiquitin E3 ligase bucket. The shared GO molecular-function target is ubiquitin protein ligase activity.
    • [class] Ubiquitin Proteasome System|E3 ubiquitin and UBL ligases
      status=context_only scope=too_broad_to_propagate GO=[GO:0061630 ubiquitin protein ligase activity]
      rationale: This class is a genuine E3-ligase context, but its descendants include catalytic ligases, cullin scaffolds, substrate receptors, adaptors, cofactors, regulators, and UBL modifier systems. A class-level propagation would over-annotate.
    • [branch] Ubiquitin Proteasome System
      status=no_mapping scope= GO=[]
      rationale: Reviewed as the top-level UPS branch. It is a project taxonomy umbrella rather than a direct GO assertion; UPS propagation must come from manually curated child nodes.

PN row 3: Ubiquitin Proteasome System | Ubiquitin and UBL binding | E3 ligase | RING / TRAC-1 | UIM

  • UniProt: Q96A37
  • In branches: ALP, UPS
  • Signature domains: PMID: 17990982
  • Auxiliary domains: IPR001841
  • PN references (titles):
    • 17990982
  • PN-node mapping records (path + ancestors):
    • [subtype] Ubiquitin Proteasome System|Ubiquitin and UBL binding|E3 ligase|RING / TRAC-1|UIM
      status=no_mapping scope= GO=[]
      rationale: Reviewed as a narrower enzyme-family, domain, or architecture subdivision already covered by a curated parent enzyme mapping. No additional direct GO mapping is needed at this node.
    • [type] Ubiquitin Proteasome System|Ubiquitin and UBL binding|E3 ligase|RING / TRAC-1
      status=no_mapping scope= GO=[]
      rationale: Reviewed as a narrower enzyme-family, domain, or architecture subdivision already covered by a curated parent enzyme mapping. No additional direct GO mapping is needed at this node.
    • [group] Ubiquitin Proteasome System|Ubiquitin and UBL binding|E3 ligase
      status=mapped scope=ok_for_propagation_to_go GO=[GO:0061630 ubiquitin protein ligase activity]
      rationale: This PN group captures ubiquitin/UBL-binding factors that are E3 ligases. The shared molecular-function target is ubiquitin protein ligase activity.
    • [class] Ubiquitin Proteasome System|Ubiquitin and UBL binding
      status=context_only scope=too_broad_to_propagate GO=[GO:0140036 ubiquitin-modified protein reader activity]
      rationale: This class records ubiquitin/UBL-reader context, but the subtree mixes ubiquitin, SUMO, UBL-domain, domain-architecture, catalytic, signaling, trafficking, and nucleic-acid process buckets. It is useful context, not a safe direct propagation.
    • [branch] Ubiquitin Proteasome System
      status=no_mapping scope= GO=[]
      rationale: Reviewed as the top-level UPS branch. It is a project taxonomy umbrella rather than a direct GO assertion; UPS propagation must come from manually curated child nodes.

Projected GO annotations (3)

  • GO:0098792 xenophagy | scope=ok_for_propagation_to_go | goa_status=new_to_goa | from=Autophagy-Lysosome Pathway|Autophagy substrate selection|Marking substrates for selective autophagy|Xenophagy
  • GO:0061630 ubiquitin protein ligase activity | scope=ok_for_propagation_to_go | goa_status=already_in_goa_exact | from=Ubiquitin Proteasome System|E3 ubiquitin and UBL ligases|RING
  • GO:0061630 ubiquitin protein ligase activity | scope=ok_for_propagation_to_go | goa_status=already_in_goa_exact | from=Ubiquitin Proteasome System|Ubiquitin and UBL binding|E3 ligase

Note

This file is generated from the current PROTEOSTASIS phase-1 dossier and local gene-review artifacts. Edit the source review, PN mapping, or dossier rather than this generated note when correcting the underlying curation.

πŸ“„ View Raw YAML

 
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-Ξ² 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-Ξ² 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-Ξ² 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.