id: Q9BT09 gene_symbol: CNPY3 product_type: PROTEIN status: COMPLETE taxon: id: NCBITaxon:9606 label: Homo sapiens description: >- CNPY3 (Protein canopy homolog 3; also known as PRAT4A, Protein Associated with TLR4) is an endoplasmic reticulum (ER)-resident, glycosylated protein of the canopy family. It has a cleaved N-terminal signal peptide and a single saposin-like / MD-2-related lipid-recognition (ML) domain (a Saposin B-type fold stabilized by three intramolecular disulfide bonds). CNPY3 functions as a Toll-like receptor (TLR)-specific co-chaperone that works together with the ER HSP90 paralog HSP90B1 (gp96 / GRP94 / endoplasmin). The CNPY3-HSP90B1 module mediates the folding and ER-to-surface/endosome trafficking of multiple TLRs, including the cell-surface receptors TLR1, TLR2, TLR4 and TLR5 and the endosomal nucleic-acid-sensing receptors TLR7 and TLR9, but not TLR3, which is CNPY3-independent. CNPY3 is therefore required for TLR exit from the ER and for innate immune signaling downstream of these receptors. The interaction between CNPY3 and HSP90B1 is disrupted by ATP, consistent with a HSP90-co-chaperone client-handoff cycle. Biallelic loss-of-function variants in CNPY3 cause autosomal recessive developmental and epileptic encephalopathy 60 (DEE60). alternative_products: - name: '1' id: Q9BT09-1 - name: '2' id: Q9BT09-2 sequence_note: VSP_030132, VSP_030133 existing_annotations: - term: id: GO:0005102 label: signaling receptor binding evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: enables review: summary: >- Phylogenetic (IBA) annotation that CNPY3 enables signaling receptor binding. CNPY3 does physically engage the ectodomains of multiple TLRs in the ER as part of its folding/trafficking chaperone activity, so binding to these signaling receptors is supported. However, this term captures the client-engagement aspect but not the core chaperone/co-chaperone activity, and it does not distinguish CNPY3 from a true signaling effector. action: KEEP_AS_NON_CORE reason: >- CNPY3 binds TLR clients (TLR1/2/4/9) in the ER, so signaling receptor binding is defensible, but it is a downstream consequence of the chaperone function rather than the core molecular function. The more informative MF terms are unfolded protein binding and Hsp90 protein binding. Keep as non-core. supported_by: - reference_id: file:human/CNPY3/CNPY3-uniprot.txt supporting_text: >- Interacts with HSP90B1; this interaction is disrupted in the presence of ATP. Interacts with TLR1, TLR2, TLR4 and TLR9. Strongest interaction with TLR4. - term: id: GO:0005783 label: endoplasmic reticulum evidence_type: IEA original_reference_id: GO_REF:0000044 qualifier: located_in review: summary: >- IEA annotation mapping the UniProt subcellular location (Endoplasmic reticulum) to GO. CNPY3 is an ER-resident protein with a signal peptide and an ER-luminal co-chaperone function. action: ACCEPT reason: >- ER localization is well established and central to CNPY3 function as an ER co-chaperone. The more specific term endoplasmic reticulum lumen (annotated separately) is also correct. supported_by: - reference_id: file:human/CNPY3/CNPY3-uniprot.txt supporting_text: 'SUBCELLULAR LOCATION: Endoplasmic reticulum.' - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:28514442 qualifier: enables review: summary: >- Bare "protein binding" annotation from a large-scale affinity purification-mass spectrometry interactome screen (BioPlex). The WITH/FROM partner (SLITRK4) is not a functionally relevant CNPY3 client. action: MARK_AS_OVER_ANNOTATED reason: >- Per curation guidelines, bare protein binding is uninformative and does not describe the molecular function of CNPY3. This interaction derives from a high-throughput proteomics dataset with no functional follow-up and an implausible partner; it should not be treated as core. supported_by: - reference_id: PMID:28514442 supporting_text: >- BioPlex 2.0 ... uses robust affinity purification-mass spectrometry methodology to elucidate protein interaction networks ... With more than 56,000 candidate interactions. - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:32296183 qualifier: enables review: summary: >- Bare "protein binding" annotation from the HuRI binary (yeast two-hybrid) interactome map. Partners (CLDN5, KCNJ6, FAM209A, GOLM1) are not functionally relevant CNPY3 clients. action: MARK_AS_OVER_ANNOTATED reason: >- Uninformative bare protein binding from a systematic all-by-all binary interactome screen, without functional characterization. Avoid bare protein binding per curation guidelines. supported_by: - reference_id: PMID:32296183 supporting_text: >- Here we present a human 'all-by-all' reference interactome map of human binary protein interactions, or 'HuRI'. With approximately 53,000 protein-protein interactions. - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:32814053 qualifier: enables review: summary: >- Bare "protein binding" annotation from a neurodegenerative-disease interactome mapping study. Partners (DMWD, WFS1, GRN, SPRED1, ATN1, KLK6, RNF11) are not functionally relevant CNPY3 clients. action: MARK_AS_OVER_ANNOTATED reason: >- Uninformative bare protein binding from a high-throughput interactome screen, without functional follow-up. Avoid bare protein binding per curation guidelines. supported_by: - reference_id: PMID:32814053 supporting_text: >- Interactome Mapping Provides a Network of Neurodegenerative Disease Proteins and Uncovers Widespread Protein Aggregation in Affected Brains. - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:33961781 qualifier: enables review: summary: >- Bare "protein binding" annotation from BioPlex 3.0 (cell-specific AP-MS interactome). The WITH/FROM partner (SLITRK4) is not a functionally relevant CNPY3 client. action: MARK_AS_OVER_ANNOTATED reason: >- Uninformative bare protein binding from a high-throughput proteomics dataset, without functional characterization. Avoid bare protein binding per curation guidelines. supported_by: - reference_id: PMID:33961781 supporting_text: >- Dual proteome-scale networks reveal cell-specific remodeling of the human interactome. - term: id: GO:0005102 label: signaling receptor binding evidence_type: IEA original_reference_id: GO_REF:0000107 qualifier: enables review: summary: >- IEA annotation transferred from the mouse ortholog (Cnpy3/Prat4A, Q9DAU1) via Ensembl Compara. Redundant with the IBA signaling receptor binding annotation; reflects CNPY3 engagement of TLR clients. action: KEEP_AS_NON_CORE reason: >- Same rationale as the IBA signaling receptor binding annotation: it captures TLR client engagement but not the core chaperone/co-chaperone molecular function. Keep as non-core. supported_by: - reference_id: file:human/CNPY3/CNPY3-uniprot.txt supporting_text: >- Toll-like receptor (TLR)-specific co-chaperone for HSP90B1 ... Interacts with TLR1, TLR2, TLR4 and TLR9. - term: id: GO:0005788 label: endoplasmic reticulum lumen evidence_type: TAS original_reference_id: Reactome:R-HSA-1678923 qualifier: located_in review: summary: >- TAS annotation from Reactome (event "TLR folding by chaperones GP96 and CNPY3") placing CNPY3 in the ER lumen, where it acts as a co-chaperone with HSP90B1/gp96. Consistent with the signal-peptide-cleaved, ER-luminal topology of CNPY3. action: ACCEPT reason: >- ER lumen is the correct and most specific subcellular location for CNPY3 and is central to its co-chaperone function in TLR folding. supported_by: - reference_id: file:human/CNPY3/CNPY3-uniprot.txt supporting_text: >- Reactome; R-HSA-1679131; Trafficking and processing of endosomal TLR. - term: id: GO:0005788 label: endoplasmic reticulum lumen evidence_type: TAS original_reference_id: Reactome:R-HSA-1678944 qualifier: located_in review: summary: >- TAS annotation from Reactome (event "Folded full-length TLR7/8/9 dissociates from the GP96:CNPY3 complex") placing CNPY3 in the ER lumen as part of the GP96:CNPY3 TLR-folding complex. action: ACCEPT reason: >- Duplicates the ER lumen localization, which is correct and central to CNPY3 function. The Reactome event directly describes the CNPY3:GP96 co-chaperone complex acting on TLR7/8/9. supported_by: - reference_id: file:human/CNPY3/CNPY3-uniprot.txt supporting_text: >- Reactome; R-HSA-1679131; Trafficking and processing of endosomal TLR. - term: id: GO:0051082 label: unfolded protein binding evidence_type: IC original_reference_id: file:human/CNPY3/CNPY3-uniprot.txt qualifier: enables review: summary: >- Proposed new annotation capturing CNPY3's core chaperone activity: as a TLR-specific co-chaperone it binds unfolded/nascent TLR clients in the ER lumen to assist their folding. action: NEW reason: >- The curated GOA does not represent CNPY3's chaperone molecular function. Unfolded protein binding is the most appropriate MF term for its client-binding activity. supported_by: - reference_id: file:human/CNPY3/CNPY3-uniprot.txt supporting_text: >- Toll-like receptor (TLR)-specific co-chaperone for HSP90B1. Required for proper TLR folding, except that of TLR3. - term: id: GO:0051879 label: Hsp90 protein binding evidence_type: IC original_reference_id: file:human/CNPY3/CNPY3-uniprot.txt qualifier: enables review: summary: >- Proposed new annotation: CNPY3 binds the ER HSP90 paralog HSP90B1/gp96 directly as a co-chaperone; the interaction is ATP-sensitive. action: NEW reason: >- Hsp90 protein binding is the most specific MF term for the well-documented CNPY3-HSP90B1 co-chaperone interaction, which is missing from GOA. supported_by: - reference_id: file:human/CNPY3/CNPY3-uniprot.txt supporting_text: >- Interacts with HSP90B1; this interaction is disrupted in the presence of ATP. - term: id: GO:0034975 label: protein folding in endoplasmic reticulum evidence_type: IC original_reference_id: file:human/CNPY3/CNPY3-uniprot.txt qualifier: involved_in review: summary: >- Proposed new annotation for the biological process in which CNPY3 acts: assisting folding of TLRs in the ER lumen. action: NEW reason: >- The process by which CNPY3 functions (TLR folding in the ER) is not annotated. GO:0034975 captures this directly. supported_by: - reference_id: file:human/CNPY3/CNPY3-uniprot.txt supporting_text: >- Required for proper TLR folding ... and hence controls TLR exit from the endoplasmic reticulum. - term: id: GO:0045087 label: innate immune response evidence_type: IC original_reference_id: file:human/CNPY3/CNPY3-uniprot.txt qualifier: involved_in review: summary: >- Proposed new annotation: by enabling TLR folding and ER exit, CNPY3 is required for innate immune signaling. Supported by the UniProt Immunity/Innate immunity keywords and the GO:0045087 InterPro/keyword annotation in UniProt. action: NEW reason: >- CNPY3 is required for innate immune responses via its role in TLR maturation; this BP is supported but not present in the curated GOA block. supported_by: - reference_id: file:human/CNPY3/CNPY3-uniprot.txt supporting_text: >- Consequently, required for both innate and adaptive immune responses. - term: id: GO:0034123 label: positive regulation of toll-like receptor signaling pathway evidence_type: IC original_reference_id: file:human/CNPY3/CNPY3-uniprot.txt qualifier: involved_in review: summary: Proposed annotation not present in the current GOA for CNPY3. action: NEW reason: >- By enabling TLR folding and ER exit, CNPY3 is required for TLR signaling; GO:0034123 captures its positive-regulatory role in this innate immune pathway. core_functions: - description: >- ER-resident, TLR-specific co-chaperone that binds the ER HSP90 paralog HSP90B1 (gp96/GRP94) and, together with it, recognizes and assists the folding of unfolded/nascent Toll-like receptors in the ER lumen. molecular_function: id: GO:0051082 label: unfolded protein binding supported_by: - reference_id: file:human/CNPY3/CNPY3-uniprot.txt supporting_text: >- Toll-like receptor (TLR)-specific co-chaperone for HSP90B1. Required for proper TLR folding, except that of TLR3. - description: >- Binds the ER HSP90 chaperone HSP90B1 (gp96) directly as a substrate-specific co-chaperone; the interaction is ATP-sensitive, consistent with a HSP90 client-handoff cycle. molecular_function: id: GO:0051879 label: Hsp90 protein binding supported_by: - reference_id: file:human/CNPY3/CNPY3-uniprot.txt supporting_text: >- Interacts with HSP90B1; this interaction is disrupted in the presence of ATP. - description: >- Promotes folding of TLRs in the ER and controls their exit from the ER to the cell surface (TLR1/2/4/5) or endosomes (TLR7/9), thereby enabling innate immune signaling. TLR3 folding is CNPY3-independent. directly_involved_in: - id: GO:0034975 label: protein folding in endoplasmic reticulum - id: GO:0045087 label: innate immune response locations: - id: GO:0005788 label: endoplasmic reticulum lumen supported_by: - reference_id: file:human/CNPY3/CNPY3-uniprot.txt supporting_text: >- Required for proper TLR folding ... and hence controls TLR exit from the endoplasmic reticulum. Consequently, required for both innate and adaptive immune responses. proposed_new_terms: [] suggested_questions: - question: Does human CNPY3 directly bind the same TLR set (TLR1/2/4/5/7/9 but not TLR3) as established for mouse PRAT4A, and which TLR ectodomain regions are engaged by the saposin-like/ML domain? - question: Is CNPY3 a general ER co-chaperone with additional non-TLR clients, or is its client repertoire restricted to TLRs? - question: How does the DEE60-associated pathology relate to CNPY3 chaperone function - is it driven by impaired TLR trafficking/innate immunity, or by a separate neuronal client? suggested_experiments: - description: Co-immunoprecipitation and proximity-labeling (e.g. BioID/APEX) of endogenous CNPY3 in human immune cells to define its direct clients and confirm ATP-sensitive HSP90B1 binding. - description: CNPY3 knockout/knockdown in human cells followed by flow cytometry and surface/endosomal trafficking assays for TLR1/2/4/5/7/9 (and TLR3 as a negative control) to quantify TLR maturation and ER exit. - description: Functional reconstitution of DEE60 patient missense variants (e.g. Gly125Arg) to test effects on CNPY3 stability, HSP90B1 binding, and TLR folding/trafficking. references: - id: GO_REF:0000033 title: Annotation inferences using phylogenetic trees 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: GO_REF:0000107 title: Automatic transfer of experimentally verified manual GO annotation data to orthologs using Ensembl Compara findings: [] - id: PMID:28514442 title: Architecture of the human interactome defines protein communities and disease networks. findings: - statement: >- Large-scale AP-MS interactome (BioPlex 2.0); reports a CNPY3 interaction with SLITRK4 as an uncharacterized high-throughput hit, not a functionally validated client. reference_section_type: ABSTRACT - id: PMID:32296183 title: A reference map of the human binary protein interactome. findings: - statement: >- HuRI binary (Y2H) interactome map; CNPY3 interactions reported are systematic screen hits without functional characterization. reference_section_type: ABSTRACT - id: PMID:32814053 title: Interactome Mapping Provides a Network of Neurodegenerative Disease Proteins and Uncovers Widespread Protein Aggregation in Affected Brains. findings: - statement: >- Neurodegenerative-disease interactome mapping; CNPY3 interactions are high-throughput screen hits, not functionally relevant chaperone clients. reference_section_type: TITLE - id: PMID:33961781 title: Dual proteome-scale networks reveal cell-specific remodeling of the human interactome. findings: - statement: >- BioPlex 3.0 cell-specific AP-MS interactome; CNPY3 interaction reported is a high-throughput screen hit without functional follow-up. reference_section_type: TITLE - id: Reactome:R-HSA-1678923 title: TLR folding by chaperones GP96 and CNPY3 findings: - statement: >- CNPY3 acts in the ER lumen with GP96 (HSP90B1) to fold Toll-like receptors. reference_section_type: OTHER - id: Reactome:R-HSA-1678944 title: 'Folded full-length TLR7/8/9 dissociates from the GP96:CNPY3 complex ' findings: - statement: >- Folded full-length TLR7/8/9 dissociates from the GP96:CNPY3 co-chaperone complex in the ER. reference_section_type: OTHER - id: file:human/CNPY3/CNPY3-uniprot.txt title: UniProtKB entry Q9BT09 (CNPY3_HUMAN) findings: - statement: >- Toll-like receptor (TLR)-specific co-chaperone for HSP90B1; required for proper TLR folding (except TLR3) and controls TLR exit from the ER; required for innate and adaptive immune responses. ER-localized; interacts with HSP90B1 (ATP-disrupted) and with TLR1/2/4/9 (strongest with TLR4). reference_section_type: OTHER