id: Q7Z4N8 gene_symbol: P4HA3 product_type: PROTEIN status: COMPLETE taxon: id: NCBITaxon:9606 label: Homo sapiens description: P4HA3 (prolyl 4-hydroxylase subunit alpha-3) is the third catalytic alpha-subunit isoform of collagen prolyl 4-hydroxylase (C-P4H). The active enzyme is an alpha2-beta2 heterotetramer in which the two catalytic alpha subunits combine with two beta subunits that are identical to protein disulfide isomerase (PDI/P4HB). P4HA3 is an endoplasmic reticulum lumenal, Fe(II)- and 2-oxoglutarate-dependent dioxygenase that, using L-ascorbate as a cofactor and molecular oxygen as co-substrate, hydroxylates proline residues in -X-Pro-Gly- triplets of procollagen and related proteins to form trans-4-hydroxyproline (consuming 2-oxoglutarate and producing succinate and CO2). 4-hydroxyproline formation is essential for folding and thermal stability of the collagen triple helix. The alpha-3 isoenzyme has catalytic properties similar to the type I and type II C-P4Hs but with intermediate peptide-substrate binding properties, and its mRNA is expressed broadly but at much lower levels than the alpha(I)/alpha(II) isoforms, being most abundant in placenta, liver, and fetal skin and detectable in vascular smooth muscle and the fibrous cap of atherosclerotic lesions. alternative_products: - name: '1' id: Q7Z4N8-1 - name: '2' id: Q7Z4N8-2 sequence_note: VSP_031146, VSP_031147 - name: '3' id: Q7Z4N8-3 sequence_note: VSP_054131 existing_annotations: - term: id: GO:0004656 label: procollagen-proline 4-dioxygenase activity evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: enables review: summary: Phylogenetic (IBA) assignment of the core collagen prolyl 4-hydroxylase activity, consistent with the direct experimental EXP evidence for P4HA3 and with conserved catalytic residues across the P4HA family. action: ACCEPT reason: This is the defining core molecular function of P4HA3; the IBA inference agrees with the experimental characterization of the recombinant alpha(III) enzyme. supported_by: - reference_id: PMID:14500733 supporting_text: led to the formation of an active enzyme that hydroxylated collagen chains and a collagen-like peptide and appeared to be an [alpha(III)]2 beta 2 tetramer - term: id: GO:0016222 label: procollagen-proline 4-dioxygenase complex evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: part_of review: summary: P4HA3 is the catalytic alpha subunit of the alpha2-beta2 collagen prolyl 4-hydroxylase tetramer, in which the beta subunit is PDI. Membership in the prolyl 4-hydroxylase complex is the correct cellular-component assignment. action: ACCEPT reason: Directly supported; the recombinant alpha(III) assembles with PDI into an [alpha(III)]2-beta2 tetramer, i.e. the procollagen-proline 4-dioxygenase complex. supported_by: - reference_id: PMID:14500733 supporting_text: The vertebrate enzymes are alpha 2 beta 2 tetramers, the beta-subunit being identical to protein-disulfide isomerase (PDI) - term: id: GO:0004656 label: procollagen-proline 4-dioxygenase activity evidence_type: IEA original_reference_id: GO_REF:0000120 qualifier: enables review: summary: Electronic assignment of the core dioxygenase activity from the EC/Rhea mapping (EC 1.14.11.2; RHEA:18945), redundant with the experimental and IBA evidence. action: ACCEPT reason: Correct core molecular function; the EC/Rhea reaction matches the curated catalytic activity of P4HA3. supported_by: - reference_id: file:human/P4HA3/P4HA3-uniprot.txt supporting_text: EC=1.14.11.2 {ECO:0000269|PubMed:14500733} - term: id: GO:0005506 label: iron ion binding evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables review: summary: P4HA3 binds one Fe(2+) ion per subunit as the catalytic metal of its Fe(II)/2-oxoglutarate-dependent dioxygenase reaction; the InterPro-based electronic assignment is accurate. action: ACCEPT reason: Iron coordination (residues 440, 442, 510) is essential for catalysis; correct cofactor molecular function supporting the core activity. supported_by: - reference_id: file:human/P4HA3/P4HA3-uniprot.txt supporting_text: Binds 1 Fe(2+) ion per subunit - term: id: GO:0005783 label: endoplasmic reticulum evidence_type: IEA original_reference_id: GO_REF:0000120 qualifier: located_in review: summary: Collagen prolyl 4-hydroxylation occurs in the ER; the electronic ER localization is consistent with the UniProt subcellular location and the secretory-pathway signal peptide. action: ACCEPT reason: Correct compartment; the more specific ER lumen term is also annotated. supported_by: - reference_id: file:human/P4HA3/P4HA3-uniprot.txt supporting_text: 'SUBCELLULAR LOCATION: Endoplasmic reticulum lumen' - term: id: GO:0005788 label: endoplasmic reticulum lumen evidence_type: IEA original_reference_id: GO_REF:0000044 qualifier: located_in review: summary: The C-P4H tetramer resides and acts in the ER lumen, the site of procollagen folding; this is the precise correct compartment for P4HA3. action: ACCEPT reason: Correct and specific subcellular location, supported by the UniProt subcellular location and Reactome curation. supported_by: - reference_id: file:human/P4HA3/P4HA3-uniprot.txt supporting_text: 'SUBCELLULAR LOCATION: Endoplasmic reticulum lumen' - term: id: GO:0016705 label: oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables review: summary: P4HA3 is a 2-oxoglutarate/Fe(II)-dependent dioxygenase that incorporates oxygen using paired donors (prolyl substrate and 2-oxoglutarate); this is a correct parent molecular function of the specific procollagen-proline 4-dioxygenase activity. action: ACCEPT reason: Accurate general oxidoreductase chemistry; the specific GO:0004656 captures the core function more informatively, but this parent term is not wrong. supported_by: - reference_id: file:human/P4HA3/P4HA3-uniprot.txt supporting_text: Reaction=L-prolyl-[collagen] + 2-oxoglutarate + O2 = trans-4-hydroxy-L- prolyl-[collagen] + succinate + CO2 - term: id: GO:0031418 label: L-ascorbic acid binding evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables review: summary: L-ascorbate (vitamin C) is a required cofactor of collagen prolyl 4-hydroxylases, maintaining the iron in the reduced state; the electronic assignment is accurate (KM 370 uM for L-ascorbate). action: ACCEPT reason: Correct cofactor-binding molecular function supporting the core dioxygenase activity. supported_by: - reference_id: file:human/P4HA3/P4HA3-uniprot.txt supporting_text: Name=L-ascorbate - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:25416956 qualifier: enables review: summary: Bare protein binding derived from a large-scale binary (Y2H) interactome reference map. The captured partners are systematic-screen hits unrelated to the physiological PDI/collagen interactions, and the term is uninformative. action: KEEP_AS_NON_CORE reason: Real IntAct interactome captures but uninformative bare protein binding; not the functional alpha2-beta2/PDI interaction, so not elevated to core (per curation guidance). supported_by: - reference_id: PMID:25416956 supporting_text: a systematic map of ?14,000 high-quality human binary protein-protein interactions - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:26871637 qualifier: enables review: summary: Bare protein binding from a high-throughput interactome screen examining alternative splicing isoform interactions; uninformative for P4HA3's catalytic function. action: KEEP_AS_NON_CORE reason: Valid high-throughput interaction capture but uninformative bare protein binding; does not reflect the core collagen-hydroxylase function. supported_by: - reference_id: file:human/P4HA3/P4HA3-uniprot.txt supporting_text: 'Q7Z4N8; O43379: WDR62' - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:31515488 qualifier: enables review: summary: Bare protein binding from a high-throughput interactome screen assaying disruption of protein interactions by genetic variants; partners are screen captures unrelated to the core enzymatic function. action: KEEP_AS_NON_CORE reason: Real interactome capture but uninformative bare protein binding; not a core function. supported_by: - reference_id: file:human/P4HA3/P4HA3-uniprot.txt supporting_text: 'Q7Z4N8; P43356: MAGEA2B' - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:32296183 qualifier: enables review: summary: Bare protein binding from the HuRI binary interactome reference map; the large partner list comprises systematic Y2H captures (keratin-associated proteins, MAGE proteins, transcription factors, etc.) not relevant to collagen prolyl 4-hydroxylation. action: KEEP_AS_NON_CORE reason: Real interactome captures but uninformative bare protein binding; per guidelines, kept non-core rather than elevated or removed. supported_by: - reference_id: file:human/P4HA3/P4HA3-uniprot.txt supporting_text: 'Q7Z4N8; O14964: HGS' - term: id: GO:0004656 label: procollagen-proline 4-dioxygenase activity evidence_type: EXP original_reference_id: PMID:14500733 qualifier: enables review: summary: Direct experimental demonstration that recombinant human alpha(III), coexpressed with PDI, forms an active enzyme that hydroxylates collagen chains and a collagen-like peptide. This is the founding biochemical characterization establishing P4HA3's core function. action: ACCEPT reason: Core molecular function with direct experimental (EXP) support; the recombinant enzyme hydroxylated collagen substrates with catalytic properties similar to the type I/II C-P4Hs. supported_by: - reference_id: PMID:14500733 supporting_text: led to the formation of an active enzyme that hydroxylated collagen chains and a collagen-like peptide and appeared to be an [alpha(III)]2 beta 2 tetramer - term: id: GO:0005788 label: endoplasmic reticulum lumen evidence_type: TAS original_reference_id: Reactome:R-HSA-1650808 qualifier: located_in review: summary: Reactome curation of P4HA3 in the ER lumen, where the collagen prolyl 4-hydroxylase reaction occurs; consistent with the UniProt subcellular location. action: ACCEPT reason: Correct and specific compartment; redundant with the IEA ER lumen annotation. supported_by: - reference_id: file:human/P4HA3/P4HA3-uniprot.txt supporting_text: 'SUBCELLULAR LOCATION: Endoplasmic reticulum lumen' - term: id: GO:0005788 label: endoplasmic reticulum lumen evidence_type: TAS original_reference_id: Reactome:R-HSA-9918779 qualifier: located_in review: summary: Reactome curation of ER-lumen localization in the context of proline hydroxylation of a (viral) polyprotein substrate; the compartment assignment for P4HA3 is correct. action: ACCEPT reason: Correct compartment; redundant with other ER lumen annotations. supported_by: - reference_id: file:human/P4HA3/P4HA3-uniprot.txt supporting_text: 'SUBCELLULAR LOCATION: Endoplasmic reticulum lumen' references: - id: GO_REF:0000002 title: Gene Ontology annotation through association of InterPro records with GO terms findings: [] - 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:0000120 title: Combined Automated Annotation using Multiple IEA Methods findings: [] - id: PMID:14500733 title: Identification and characterization of a third human, rat, and mouse collagen prolyl 4-hydroxylase isoenzyme. findings: - statement: Cloning and characterization of the third vertebrate C-P4H alpha-subunit isoform (alpha-III); recombinant human alpha(III) coexpressed with PDI forms an active [alpha(III)]2-beta2 tetramer that hydroxylates collagen chains and a collagen-like peptide. reference_section_type: ABSTRACT - statement: Catalytic properties of the alpha(III) C-P4H are very similar to the type I and II C-P4Hs, with peptide-binding properties intermediate between the type I and type II enzymes; alpha(III) mRNA is expressed broadly but at much lower levels than alpha(I)/alpha(II). reference_section_type: ABSTRACT reference_review: relevance: HIGH correctness: VERIFIED review_notes: Founding biochemical characterization of P4HA3. Cache is abstract-only, but the core MF (procollagen-proline 4-dioxygenase activity, EC 1.14.11.2 / RHEA:18945) and the alpha2-beta2(PDI) tetramer are directly supported by the abstract and anchor the EXP GOA annotation. - id: PMID:25416956 title: A proteome-scale map of the human interactome network. findings: - statement: Systematic high-quality binary (Y2H) interactome map of ~14,000 human protein-protein interactions; source of P4HA3 bare protein binding (IPI) annotations. reference_section_type: ABSTRACT reference_review: relevance: LOW correctness: VERIFIED review_notes: High-throughput binary interactome screen; provides bare protein binding captures only, not informative for P4HA3's catalytic function. - id: PMID:26871637 title: Widespread Expansion of Protein Interaction Capabilities by Alternative Splicing. findings: - statement: High-throughput Y2H interactome of alternative-splice isoforms; source of P4HA3 bare protein binding (IPI) annotation. reference_section_type: ABSTRACT reference_review: relevance: LOW correctness: VERIFIED review_notes: Isoform interactome screen; uninformative bare protein binding for P4HA3. - id: PMID:31515488 title: Extensive disruption of protein interactions by genetic variants across the allele frequency spectrum in human populations. findings: - statement: Y2H interactome assaying perturbation of protein-protein interactions by human genetic variants; source of P4HA3 bare protein binding (IPI) annotation. reference_section_type: ABSTRACT reference_review: relevance: LOW correctness: VERIFIED review_notes: Variant-perturbation interactome screen; uninformative bare protein binding. - id: PMID:32296183 title: A reference map of the human binary protein interactome. findings: - statement: HuRI, an all-by-all reference map of ~53,000 human binary protein-protein interactions; source of the largest set of P4HA3 bare protein binding (IPI) captures. reference_section_type: ABSTRACT reference_review: relevance: LOW correctness: VERIFIED review_notes: HuRI binary interactome reference map; uninformative bare protein binding for P4HA3's function. - id: PMID:34009234 title: Collagen prolyl 4-hydroxylases modify tumor progression. findings: [] reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: 'PubMed-verified review (Shi et al., Acta Biochim Biophys Sin 2021; DOI 10.1093/abbs/gmab065). Reviews the three C-P4H isoenzymes (P4HA1/2/3, alpha2-beta2 with single P4HB), their regulation (cytokines, transcription factors, miRNAs), overexpression in tumors, and C-P4H inhibitors as anti-tumor strategies. Supports the core alpha2-beta2/PDI framing and the (non-core) tumor-progression context for P4HA3.' - id: PMID:36588128 title: P4HA3 promotes clear cell renal cell carcinoma progression via the PI3K/AKT/GSK3beta pathway. findings: [] reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: 'PubMed-verified (Zhang et al., Med Oncol 2023; DOI 10.1007/s12032-022-01926-2). P4HA3 is overexpressed in clear cell renal cell carcinoma (TCGA) and high expression correlates with worse overall survival; P4HA3 knockdown inhibits ccRCC cell proliferation, migration and invasion, with GSEA/Western blot implicating EMT and PI3K/AKT/GSK3beta signaling. P4HA3-specific functional-perturbation evidence in a tumor context (non-core for the catalytic annotation).' - id: PMID:39362976 title: Comprehensive analysis reveals that P4HA3 is a prognostic and diagnostic gastric cancer biomarker that can predict immunotherapy efficacy. findings: [] reference_review: relevance: LOW correctness: VERIFIED review_notes: 'PubMed-verified (Yu et al., Sci Rep 2024; DOI 10.1038/s41598-024-73784-z). TCGA/TIMER bioinformatic analysis associating elevated P4HA3 with adverse prognosis and immune-microenvironment features (immune infiltration, TMB, MSI, immune checkpoints) in gastric cancer; proposes P4HA3 as an immunotherapy-response biomarker. Correlative biomarker context, non-core.' - id: PMID:39394821 title: 'Collagen prolyl 4-hydroxylase subunit alpha member-induced head and neck squamous cell carcinoma aggressiveness is antagonized by LLGL2 via reduced expression of occludin.' findings: [] reference_review: relevance: LOW correctness: VERIFIED review_notes: 'PubMed-verified (Xu et al., Acta Biochim Biophys Sin 2024; DOI 10.3724/abbs.2024140). TCGA analysis of all three C-P4HA alpha subunits (including P4HA3) in HNSCC; combined higher expression of the three is prognostically adverse and antagonized by LLGL2 via occludin; knockdown of C-P4HA subunits reduces migration/invasion. Family-level cancer context, non-core for P4HA3.' - id: PMID:39504328 title: A computational analysis of the oncogenic and anti-tumor immunity role of P4HA3 in human cancers. findings: [] reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: 'PubMed-verified (Huang et al., PLoS Comput Biol 2024; DOI 10.1371/journal.pcbi.1012284). Pan-cancer (33 tumor types) analysis with experimental validation: P4HA3 expression correlates with tumor-microenvironment infiltration, proliferation and EMT markers; P4HA3 depletion inhibits proliferation/migration/invasion and enhances anti-tumor PD-1/PD-L1 immunotherapy response (including TNBC PDX models). P4HA3-specific functional evidence in tumor/immune context (non-core for the catalytic annotation).' - id: Reactome:R-HSA-1650808 title: Prolyl 4-hydroxylase converts collagen prolines to 4-hydroxyprolines findings: [] - id: Reactome:R-HSA-9918779 title: Proline hydroxylases hydroxylate Polyprotein findings: [] - id: file:human/P4HA3/P4HA3-uniprot.txt title: UniProt entry Q7Z4N8 (P4HA3_HUMAN), Prolyl 4-hydroxylase subunit alpha-3 findings: - statement: ER-lumenal Fe(II)/2-oxoglutarate-dependent dioxygenase (EC 1.14.11.2) that forms 4-hydroxyproline in -Xaa-Pro-Gly- sequences in collagens; binds 1 Fe(2+) per subunit and requires L-ascorbate; catalytic alpha subunit of an alpha2-beta2 tetramer with PDI. reference_section_type: OTHER core_functions: - description: Catalytic alpha subunit of collagen prolyl 4-hydroxylase that, as part of an alpha2-beta2 tetramer with PDI, hydroxylates proline residues in -X-Pro-Gly- triplets of procollagen to form trans-4-hydroxyproline, a modification required for collagen triple-helix folding and stability. molecular_function: id: GO:0004656 label: procollagen-proline 4-dioxygenase activity locations: - id: GO:0005788 label: endoplasmic reticulum lumen supported_by: - reference_id: PMID:14500733 supporting_text: led to the formation of an active enzyme that hydroxylated collagen chains and a collagen-like peptide and appeared to be an [alpha(III)]2 beta 2 tetramer - reference_id: file:human/P4HA3/P4HA3-uniprot.txt supporting_text: Catalyzes the post-translational formation of 4- - description: Fe(II)- and 2-oxoglutarate-dependent dioxygenase that uses L-ascorbate as a cofactor and molecular oxygen as co-substrate, coupling decarboxylation of 2-oxoglutarate to hydroxylation of the prolyl substrate. molecular_function: id: GO:0004656 label: procollagen-proline 4-dioxygenase activity supported_by: - reference_id: file:human/P4HA3/P4HA3-uniprot.txt supporting_text: Reaction=L-prolyl-[collagen] + 2-oxoglutarate + O2 = trans-4-hydroxy-L- prolyl-[collagen] + succinate + CO2 proposed_new_terms: [] suggested_questions: - question: Does P4HA3-containing C-P4H have a distinct substrate preference (e.g. for particular collagen types or non-collagenous Pro-Gly-containing substrates) that distinguishes it from the alpha(I)- and alpha(II)-containing isoenzymes, given its intermediate peptide-binding properties? - question: Is the elevated P4HA3 expression in vascular smooth muscle, atherosclerotic fibrous cap, and certain tumors associated with a direct collagen-hydroxylation role in ECM remodeling, or with non-catalytic/contextual functions? suggested_experiments: - description: Reconstitute recombinant [alpha(III)]2-beta2 (P4HA3/PDI) C-P4H and measure steady-state kinetics and substrate specificity against a panel of collagen-type peptides versus the type I and type II C-P4Hs to define isoenzyme-specific substrate preferences. - description: CRISPR knockout or knockdown of P4HA3 in cells with high endogenous expression (e.g. placental or vascular smooth muscle cells) followed by mass spectrometry quantification of collagen 4-hydroxyproline content to assess its non-redundant contribution to collagen hydroxylation in vivo.