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 of the ER HSP90 paralog HSP90B1 (gp96 / GRP94 / endoplasmin). Mechanistic experiments on mouse Cnpy3 established that the Cnpy3-Hsp90b1 module promotes TLR substrate loading and is required for TLR folding and ER-to-surface/endosome trafficking. Human CNPY3 is inferred to share this conserved co-chaperone mechanism. Its clients include 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).
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005102 signaling receptor binding | IBA GO_REF:0000033 | KEEP AS NON CORE | 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. 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. Hsp90 protein binding is more informative; the evidence does not demonstrate autonomous binding of unfolded protein by CNPY3. Keep as non-core. Propagation Review Root cause: NO FAILURE NON CORE Sources checked: PANTHER:PTN008355430 · PANTHER:PTN008355430 SUPPORTS TRANSFER PAINT places GO:0005102 at this node in the correct canopy family, PTHR15382, from mouse Cnpy3 and Cnpy4 evidence. Human CNPY3 belongs to PTHR15382:SF2 and lies within the annotated clade. MGI:MGI:1919279 · mouse Cnpy3 SUPPORTS TRANSFER Mouse Cnpy3 is the experimentally characterized ortholog; PMID:20865800 shows TLR9 in a complex with Cnpy3 and gp96. MGI:MGI:1913705 · mouse Cnpy4 SUPPORTS TRANSFER Mouse Cnpy4 is a canopy-family paralog contributing descendant evidence to the ancestral receptor-binding placement. Supporting Evidence: PMID:20865800 TLR9 forms a multimolecular complex with gp96 and CNPY3, and the binding of TLR9 to either molecule requires the presence of the other. |
| GO:0005783 endoplasmic reticulum | IEA GO_REF:0000044 | ACCEPT | 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. 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. Supporting Evidence: file:human/CNPY3/CNPY3-uniprot.txt SUBCELLULAR LOCATION: Endoplasmic reticulum. |
| GO:0005515 protein binding | IPI PMID:28514442 Architecture of the human interactome defines protein commun... | KEEP AS NON CORE | Summary: Bare "protein binding" annotation from a large-scale affinity purification-mass spectrometry interactome screen (BioPlex). The WITH/FROM partner is SLITRK4; the screen does not establish what biological role, if any, this interaction has for CNPY3. Reason: IPI asserts an observed physical CNPY3-SLITRK4 interaction, and no evidence contradicts that observation, so the annotation is retained. GO:0005515 provides no mechanistic interpretation and this screen does not establish a more specific MF, so the interaction remains non-core rather than invalid. Supporting Evidence: file:human/CNPY3/CNPY3-goa.tsv GO:0005515 protein binding molecular_function ECO:0000353 IPI PMID:28514442 UniProtKB:Q8IW52 |
| GO:0005515 protein binding | IPI PMID:32296183 A reference map of the human binary protein interactome. | KEEP AS NON CORE | Summary: Bare "protein binding" annotation from the HuRI binary (yeast two-hybrid) interactome map. The collapsed signature contains CLDN5, KCNJ6, FAM209A and GOLM1, but no single informative MF describes this heterogeneous set. Reason: IPI asserts observed physical interactions with this partner set, and no evidence contradicts those observations, so the annotation is retained. GO:0005515 provides no mechanistic interpretation and the heterogeneous set does not support one specific MF replacement, so it remains non-core rather than invalid. Supporting Evidence: file:human/CNPY3/CNPY3-goa.tsv GO:0005515 protein binding molecular_function ECO:0000353 IPI PMID:32296183 UniProtKB:O00501 |
| GO:0005515 protein binding | IPI PMID:32814053 Interactome Mapping Provides a Network of Neurodegenerative ... | KEEP AS NON CORE | Summary: Bare "protein binding" annotation from a neurodegenerative-disease interactome mapping study. Partners (DMWD, WFS1, GRN, SPRED1, ATN1, KLK6, RNF11) form a heterogeneous collapsed signature without a common specific MF. Reason: IPI asserts observed physical interactions with this partner set, and no evidence contradicts those observations, so the annotation is retained. GO:0005515 provides no mechanistic interpretation and the heterogeneous set does not support one specific MF replacement, so it remains non-core rather than invalid. Supporting Evidence: file:human/CNPY3/CNPY3-goa.tsv GO:0005515 protein binding molecular_function ECO:0000353 IPI PMID:32814053 UniProtKB:G5E9A7 |
| GO:0005515 protein binding | IPI PMID:33961781 Dual proteome-scale networks reveal cell-specific remodeling... | KEEP AS NON CORE | Summary: Bare "protein binding" annotation from BioPlex 3.0 (cell-specific AP-MS interactome). The WITH/FROM partner is SLITRK4; the screen does not establish what biological role, if any, this interaction has for CNPY3. Reason: IPI asserts an observed physical CNPY3-SLITRK4 interaction, and no evidence contradicts that observation, so the annotation is retained. GO:0005515 provides no mechanistic interpretation and this screen does not establish a more specific MF, so the interaction remains non-core rather than invalid. Supporting Evidence: file:human/CNPY3/CNPY3-goa.tsv GO:0005515 protein binding molecular_function ECO:0000353 IPI PMID:33961781 UniProtKB:Q8IW52 |
| GO:0005102 signaling receptor binding | IEA GO_REF:0000107 | KEEP AS NON CORE | 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. 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. Supporting Evidence: file:human/CNPY3/CNPY3-uniprot.txt Interacts with TLR1, TLR2, TLR4 and TLR9 |
| GO:0005788 endoplasmic reticulum lumen | TAS Reactome:R-HSA-1678923 | ACCEPT | 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. Reason: ER lumen is the correct and most specific subcellular location for CNPY3 and is central to its co-chaperone function in TLR folding. Supporting Evidence: file:human/CNPY3/CNPY3-uniprot.txt Reactome; R-HSA-1679131; Trafficking and processing of endosomal TLR. |
| GO:0005788 endoplasmic reticulum lumen | TAS Reactome:R-HSA-1678944 | ACCEPT | 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. 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. Supporting Evidence: file:human/CNPY3/CNPY3-uniprot.txt Reactome; R-HSA-1679131; Trafficking and processing of endosomal TLR. |
| GO:0051879 Hsp90 protein binding | IC file:human/CNPY3/CNPY3-uniprot.txt | NEW | Summary: Proposed new annotation: CNPY3 binds the ER HSP90 paralog HSP90B1/gp96 directly as a co-chaperone; the interaction is ATP-sensitive. Reason: Hsp90 protein binding is the most specific MF term for the well-documented CNPY3-HSP90B1 co-chaperone interaction, which is missing from GOA. Direct biochemical evidence is from the mouse ortholog and the conserved human interaction is reviewed by UniProt on a by-similarity basis. Supporting Evidence: PMID:20865800 gp96 directly interacts with CNPY3, and the complex dissociates in the presence of adenosine triphosphate (ATP). file:human/CNPY3/CNPY3-uniprot.txt Interacts with HSP90B1; this interaction is disrupted in the presence of ATP. |
| GO:0034975 protein folding in endoplasmic reticulum | IC file:human/CNPY3/CNPY3-uniprot.txt | NEW | Summary: Proposed new annotation for the biological process in which CNPY3 acts: assisting folding of TLRs in the ER lumen. Reason: The process by which CNPY3 functions (TLR folding in the ER) is not annotated. GO:0034975 captures this directly. Mouse knockout, interaction disruption and substrate-maturation experiments establish the mechanism; its application to human CNPY3 is orthology-supported. Supporting Evidence: PMID:20865800 Taken together, we concluded that gp96–CNPY3 interaction is critical for folding TLR9. |
| GO:0072657 protein localization to membrane | IC file:human/CNPY3/CNPY3-uniprot.txt | NEW | Summary: Proposed process annotation for CNPY3-dependent exit of folded TLR clients from the ER and their delivery to surface or endosomal membranes. Reason: GO:0072657 captures the trafficking consequence of the CNPY3-gp96 folding module: after mouse Cnpy3 silencing, TLR9 precursors remain trapped in the ER and surface TLR4 is reduced. The direct experiments are in mouse; transfer to human CNPY3 is based on conserved orthology and the reviewed human UniProt function. Supporting Evidence: PMID:20865800 we found that TLR9 precursors were trapped in the ER of cells with silenced expression of either gp96 or CNPY3 (Fig. 5b). |
| GO:0045087 innate immune response | IC file:human/CNPY3/CNPY3-uniprot.txt | NEW | 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. 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. Supporting Evidence: file:human/CNPY3/CNPY3-uniprot.txt Consequently, required for both innate and adaptive immune responses. |
| GO:0034123 positive regulation of toll-like receptor signaling pathway | IC file:human/CNPY3/CNPY3-uniprot.txt | NEW | Summary: Proposed downstream process annotation reflecting loss of TLR responses when the CNPY3-gp96 maturation module is disrupted; it does not imply that CNPY3 directly catalyzes receptor signaling. Reason: By enabling TLR folding and ER exit, CNPY3 is required for TLR signaling; GO:0034123 captures this downstream positive-regulatory context. Retain as a non-core NEW process annotation, distinct from the core co-chaperone mechanism and not as a direct signaling activity. The functional evidence is from mouse Cnpy3 and is transferred to human by orthology. Supporting Evidence: PMID:20865800 This strategy effectively silenced CNPY3, as evidenced by significant reduction of CNPY3 mRNA (Fig. 4a), cell surface TLR4 expression (Fig. 4b), as well as functional loss of multiple TLRs including TLR2, TLR4, TLR7 and TLR9 (Fig. 4c,d). |
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Download this section (compressed HTML)Q: 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?
Q: Is CNPY3 a general ER co-chaperone with additional non-TLR clients, or is its client repertoire restricted to TLRs?
Q: 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?
Experiment: 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.
Experiment: 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.
Experiment: Functional reconstitution of DEE60 patient missense variants (e.g. Gly125Arg) to test effects on CNPY3 stability, HSP90B1 binding, and TLR folding/trafficking.
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