id: MODULE:hif_oxygen_sensing title: HIF oxygen sensing — prolyl hydroxylation of HIF-alpha by PHD1-3, CRL2(VHL) ubiquitination and degradation, FIH asparaginyl hydroxylation, and HIF-alpha/ARNT dimerisation with p300/CBP; EGLN1/EGLN2/EGLN3/OS9/VHL/ELOB/ELOC/CUL2/RBX1/SAT2/HIF1AN/HIF1A/EPAS1/HIF3A/ARNT/ARNT2/EP300/CREBBP/RACK1/SAT1 description: >- Hypoxia-inducible factor (HIF) is the master transcriptional regulator of the metazoan response to low oxygen. It is a heterodimer of an oxygen-labile alpha subunit (HIF-1alpha, HIF-2alpha/EPAS1, or HIF-3alpha) and a constitutive bHLH-PAS beta subunit (ARNT, also called HIF-1beta, or its paralog ARNT2). The oxygen signal is read by two families of Fe(II)- and 2-oxoglutarate-dependent dioxygenases that use molecular O2 as a cosubstrate, so their activity falls as oxygen falls. The prolyl hydroxylases PHD1, PHD2 and PHD3 (EGLN2, EGLN1 and EGLN3) hydroxylate two prolines in the oxygen-dependent degradation domain of the alpha subunit. trans-4-Hydroxyproline creates the recognition mark for the von Hippel-Lindau protein (VHL), the substrate receptor of a cullin-2 RING ubiquitin ligase (CRL2) in which the Elongin B/C module (ELOB, ELOC) bridges VHL's VHL-box to the CUL2 scaffold and the RING subunit RBX1 transfers ubiquitin. Polyubiquitinated HIF-alpha is destroyed by the proteasome, giving the protein a half-life of minutes in oxygenated cells. In parallel, the asparaginyl hydroxylase FIH (HIF1AN) hydroxylates a conserved asparagine in the C-terminal transactivation domain, which blocks binding of the coactivators p300 (EP300) and CBP (CREBBP) and so silences the fraction of alpha subunit that escapes degradation. When oxygen is scarce both hydroxylations fail: the alpha subunit accumulates, enters the nucleus, dimerises with ARNT through its bHLH and PAS domains, binds hypoxia response elements and recruits p300/CBP to activate transcription of genes for angiogenesis, erythropoiesis, glycolysis and iron handling. Several proteins tune the switch without sensing oxygen themselves: OS-9 scaffolds HIF-1alpha with the prolyl hydroxylases, SSAT2 (SAT2) stabilises the VHL-Elongin C interaction, and RACK1 with SSAT1 (SAT1) recruits the Elongin B/C ligase to HIF-1alpha independently of hydroxylation and of VHL. Loss of VHL abolishes degradation and gives constitutive HIF activity, the basis of von Hippel-Lindau disease and of clear-cell renal carcinoma; EPAS1 and EGLN1 mutations cause erythrocytosis and paraganglioma. status: DRAFT scope: CONCRETE evidence: - source_id: PW:0000360 title: hypoxia inducible factor pathway statement: >- The module boundary follows the RGD Pathway Ontology term and its interactive diagram. The diagram centres on the hydroxylated alpha subunit (drawn as Hif1a+O2) and draws Egln1 positively affecting it and associated with Os9; Hif1a+O2 associated with Os9, Sat1, Sat2 and Vhl; Hif1an positively affecting Hif1a+O2; Hif1a+O2 negatively affecting Crebbp and EP300; Gnb2l1 (Rack1) associated with Hif1a+O2, Sat1 and Tceb1; Sat2 associated with Tceb1 and Vhl; Tceb1 associated with Vhl and with the Tceb1 recruited complex; and the deoxygenated alpha subunit (Hif1a-O2) positively affecting Hif target genes, with the labels "Hif-alpha ubiquitination, proteasomal degradation", "P-OH" and "N-OH". The record text adds the three alpha genes, the second beta subunit Arnt2, the three prolyl hydroxylases with Egln1/Phd2 as the essential one, and the oxygen-independent Rack1 route. Derived through projects/RGD_PATHWAY_ONTOLOGY (pathways/PW_0000360.yaml). - source_id: GO:0097411 title: hypoxia-inducible factor-1alpha signaling pathway statement: >- The curated narrowMatch recorded for PW:0000360 in projects/RGD_PATHWAY_ONTOLOGY/data/pw_go_curated.sssom.yaml, and this module's process concept. The mapping comment notes that GO:0097411 is HIF-1alpha only while the PW pathway also covers HIF-2alpha (EPAS1), which is why the alpha subunit is modelled as a variant set rather than as HIF1A alone. - source_id: GO:0071456 title: cellular response to hypoxia statement: >- The curated broadMatch recorded for PW:0000360 in the same SSSOM file. It is the parent process under which GO:0097411 sits (GO:0097411 part_of GO:0071456), and the process the human GO-CAM gomodel:689e7a5d00004061 places the HIF1A transcription-factor activity in. It is kept as prose context rather than as the module's core concept, since a broad cellular-response term is less informative than the signalling-pathway term. - source_id: gomodel:689e7a5d00004061 title: Regulation of Respiratory chain complex I by COXFA4L2 upon hypoxia. (Human) statement: >- The closest human GO-CAM to this module's transcriptional output: it enables HIF1A DNA-binding transcription factor activity, RNA polymerase II-specific (GO:0000981) as part of GO:0071456 cellular response to hypoxia, driving COXFA4L2 expression. No production GO-CAM models the oxygen-sensing switch itself (checked gocams/index.tsv, October 2026): there is no model containing VHL, HIF1AN, EGLN1 or EGLN2, and the only EGLN3 model (gomodel:680ad14200007201) hydroxylates GPX4 rather than a HIF alpha subunit. - source_id: gomodel:63f809ec00000611 title: The CRL2(KLHDC10) complex catalyzes ubiquitination via the DesCEND (destruction via C-end degrons) pathway (Human) statement: >- The CRL2 core of this module (CUL2 ubiquitin ligase complex scaffold activity GO:0160072, RBX1 ubiquitin protein ligase activity GO:0061630) is grounded on this curated human CRL2 model, which uses a different substrate receptor (KLHDC10 rather than VHL) and therefore grounds the scaffold and RING activities, not the substrate choice. - source_id: PMID:11292861 title: Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. statement: >- The founding result for the module: prolyl hydroxylation of the alpha subunit is the oxygen-dependent step that licenses pVHL binding, and the hydroxylase is the oxygen sensor. supporting_text: An absolute requirement for dioxygen as a cosubstrate and iron as cofactor suggests that HIF-PH functions directly as a cellular oxygen sensor. - source_id: PMID:11823643 title: Asparagine hydroxylation of the HIF transactivation domain a hypoxic switch. statement: >- Establishes the second, transcription-blocking hydroxylation as a part of the same switch, so the module has two oxygen-sensing arms rather than one. supporting_text: Full induction of HIF-1alpha and -2alpha, therefore, relies on the abrogation of both Pro and Asn hydroxylation, which during normoxia occur at the degradation and COOH-terminal transactivation domains, respectively. - source_id: PMID:10353251 title: The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. statement: >- Places pVHL in the degradation arm and shows that losing it is sufficient for constitutive HIF activity, which is why the ligase is a part of the module and not external regulation. supporting_text: In VHL-defective cells, HIF alpha-subunits are constitutively stabilized and HIF-1 is activated. - source_id: PMID:7539918 title: Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. statement: >- The heterodimer definition, and the basis for modelling the beta subunit as a shared, constitutive partner that also serves other bHLH-PAS factors. supporting_text: HIF-1 beta is a series of ARNT gene products, which can thus heterodimerize with either HIF-1 alpha or AHR. - source_id: PMID:8917528 title: An essential role for p300/CBP in the cellular response to hypoxia. statement: >- p300/CBP recruitment is the transcriptional output step that the asparaginyl hydroxylation arm switches off, so both coactivators are modelled as parts. supporting_text: Hypoxic conditions lead to the formation of a DNA binding complex containing both HIF-1 alpha and p300/CBP. notes: >- Derived from the RGD Pathway Ontology diagram PW:0000360 through the RGD_PATHWAY_ONTOLOGY project's diagram->module route. RGD curates 20 human genes to PW:0000360 (ARNT ARNT2 CREBBP CUL2 EGLN1 EGLN2 EGLN3 ELOB ELOC EP300 EPAS1 HIF1A HIF1AN HIF3A OS9 RACK1 RBX1 SAT1 SAT2 VHL). All 20 are modelled here. The mapped GO process GO:0097411 is the module's core concept and the broader GO:0071456 is kept as prose; both mappings come from the project's curated SSSOM file. The fly counterpart is modules/dmel_hif_complex.yaml, which models only the Sima-Tgo heterodimer (the FlyBase HIF gene group) and explicitly leaves the oxygen sensor Fatiga (Hph) and the VHL ligase out as regulators; this mammalian module takes the opposite boundary, because the RGD pathway is the sensing-and-degradation switch and the heterodimer is one step inside it. Nothing is duplicated between the two: the fly module is referenced here only in prose, since cross-species inclusion by module_ref would assert an identity between the fly complex and the human one that this module does not need. Molecular functions were taken from each protein's experimental GO annotations in QuickGO (checked 2026-10-28) and, where a gene review exists, cross-checked against its core functions. EGLN1, EGLN2 and EGLN3 all carry GO:0160082 hypoxia-inducible factor-proline dioxygenase activity (EGLN1 EXP/IDA and the core function of the completed EGLN1 review; EGLN2 and EGLN3 EXP), whose definition and Rhea xref RHEA:48400 are exactly the reaction this module's first step performs; the parent GO:0031545 peptidyl-proline 4-dioxygenase activity was not used for the HIF reaction, although it is the term the Reactome HIF reactions map to and the term the EGLN3 GO-CAM uses for its GPX4 substrate. HIF1AN carries GO:0036140 [protein]-asparagine 3-dioxygenase activity (IDA/IMP; RHEA:54260). VHL carries GO:1990756 ubiquitin-like ligase-substrate adaptor activity (IDA), ELOC GO:0030674 protein-macromolecule adaptor activity (IBA; core in the ELOC review), ELOB GO:0031625 ubiquitin protein ligase binding (IDA; core in the ELOB review), CUL2 GO:0160072 ubiquitin ligase complex scaffold activity (IDA) and RBX1 GO:0061630 ubiquitin protein ligase activity (IDA; core in the RBX1 review). The alpha subunits carry GO:0000981 DNA-binding transcription factor activity, RNA polymerase II-specific (HIF1A IDA, HIF3A IDA, EPAS1 IBA/ISA), the term both human HIF1A GO-CAMs use; the more specific GO:0001228 DNA-binding transcription activator activity, RNA polymerase II-specific is available for HIF1A (IDA/IMP) but not experimentally for EPAS1 or HIF3A, and HIF3A's IPAS variant is a repressor, so the shared parent is used across the variant set. ARNT and ARNT2 carry GO:0046982 protein heterodimerization activity (both IDA; core in the ARNT review) — the beta subunit's role here is to provide the dimerisation partner and the composite DNA-binding surface, and its own GO:0000981 annotation is contributes_to (IDA) rather than enables. EP300 and CREBBP carry GO:0003713 transcription coactivator activity (IDA; core in both reviews). Boundary decisions. (1) Target genes are out of scope. VEGFA and EPO are the outputs the pathway acts on, not participants: they bind no HIF, hydroxylate nothing and carry no signalling activity, exactly the substrate-versus-participant distinction CLAUDE.md requires. RGD draws them behind a single "Hif target genes" sub-page link rather than as genes, and neither is in the PW:0000360 gene list. (2) OS9, SAT2, RACK1 and SAT1 are modelled as regulatory parts rather than relegated to context, because each has a primary paper with gain- and loss-of-function evidence placing it in a physical complex with HIF-1alpha and with a named component of the degradation machinery (OS-9 with the prolyl hydroxylases, PMID:15721254; SSAT2 with VHL and Elongin C, PMID:17558023; RACK1 with Elongin C, PMID:17244529; SSAT1 with RACK1, PMID:17875644). None of the four has a GO molecular function that describes this role: OS9's only experimental MF is GO:0140032 glycosylation-dependent protein binding (IDA), from its ERAD lectin function; SAT1 and SAT2 carry only their polyamine acetyltransferase activity GO:0004145 (and SAT2 also a NOT|enables GO:0004145 IDA row, reflecting the dispute over whether it acetylates spermidine at all), which is not the activity at work here; RACK1's adaptor role is captured only by the broad GO:0060090 molecular adaptor activity (TAS; core in the RACK1 review, which already records HIF1A as a client). Their annotons therefore carry a preferred_term and evidence but no GO identifier, except RACK1 where the broad adaptor term is used, rather than asserting a plausible-looking term the ontology does not support. This is a candidate for proposed new terms once the scaffolding roles are better characterised. (3) The oxygen-independent arm (RACK1 + SAT1) is marked optional: it operates on the same substrate but is not required for the oxygen switch, and it was defined largely through HSP90 inhibition. (4) HIF3A is modelled as a third alpha variant rather than as a separate inhibitory module. Its full-length product is a hydroxylation-regulated, ARNT-dimerising transcription factor (GO:0000981 IDA, GO:0000978 IDA) that lacks a C-terminal transactivation domain, and its short IPAS splice variant is a dominant-negative that sequesters HIF-1alpha (PMID:11734856). Both readings are recorded on the annoton; splitting the locus into two modules would overstate how well the isoform repertoire is resolved. (5) HSP90, which competes with RACK1 for the PAS-A domain, is left out: it is a general chaperone whose involvement is shown mainly through inhibitor treatment, and including it would pull the HSP90 cycle into the module. The same applies to the HIF target-gene feedback on EGLN1 (PHD2 is itself HIF-inducible) and to the 2-oxoglutarate and ascorbate supply that all four dioxygenases need. (6) The Elongin B/C module is modelled as two explicit annotons, not folded into a complex descriptor, because both ELOB and ELOC have completed gene reviews whose core functions are exactly these adaptor activities, and because the same module serves CRL5 ligases elsewhere (see modules/reelin_signaling.yaml, whose CRL5(SOCS7) part omits Elongin B/C following its GO-CAM). The generic cullin-RING machinery is not re-modelled here: neddylation, the CAND1/CSN cycle and the proteasome itself are outside the boundary, and the 26S proteasome step is recorded as a process term on the ligase annotons rather than as a part. GO-CAM and GOA observations for upstream review. No production GO-CAM covers the oxygen sensor: gocams/index.tsv (October 2026) has no model containing VHL, HIF1AN, EGLN1, EGLN2, ELOB, ELOC, EPAS1, HIF3A, OS9, SAT1 or SAT2, and the two HIF1A models (gomodel:689e7a5d00004061, gomodel:689e7a5d00004412) use HIF1A only as the transcription factor downstream of COXFA4L2. This is the largest curation gap the module reveals. In GOA, GO:0097411 hypoxia-inducible factor-1alpha signaling pathway is carried in human, mouse and rat by CYBB, PDK1, PDK3, mouse Hif1a and miR-210, but by none of EGLN1-3, VHL, HIF1AN, ARNT, EP300 or CREBBP; EGLN1 and mouse Vhl instead carry the regulatory term GO:1902072 negative regulation of hypoxia-inducible factor-1alpha signaling pathway, which is the convention this module follows (the hydroxylase and the ligase regulate the pathway rather than being part of it), so no NEW pathway annotations are proposed for them here. SAT2's human GOA carries both enables and NOT|enables GO:0004145 by IDA from different references, which is worth an upstream look. Gene reviews exist for ARNT, EGLN1, ELOB, ELOC, RBX1, EP300, CREBBP, RACK1 and SAT1; HIF1A, EPAS1, HIF3A, ARNT2, VHL, EGLN2, EGLN3, HIF1AN, CUL2, OS9 and SAT2 have none, and PANTHER family ids and PTNs are deliberately not asserted. module: id: hif_oxygen_sensing label: HIF oxygen-sensing switch module_type: SIGNALING_PATHWAY concepts: - preferred_term: hypoxia-inducible factor-1alpha signaling pathway term: id: GO:0097411 label: hypoxia-inducible factor-1alpha signaling pathway description: >- The signalling pathway mapped to PW:0000360. The GO term names HIF-1alpha, while this module covers all three alpha subunits; the extra coverage is recorded in the alpha variant set rather than by using a broader term. context: taxa: - preferred_term: Homo sapiens term: id: NCBITaxon:9606 label: Homo sapiens cellular_components: - preferred_term: cytosol term: id: GO:0005829 label: cytosol description: >- Where the prolyl hydroxylases (PHD2 is predominantly cytoplasmic) and the CRL2(VHL) ligase act on the alpha subunit. - preferred_term: nucleus term: id: GO:0005634 label: nucleus description: >- Where the stabilised alpha subunit dimerises with ARNT, binds hypoxia response elements and recruits p300/CBP. Both compartments are listed because the switch is spatially split, not because one is a refinement of the other. parts: - order: 1 role: oxygen-dependent prolyl hydroxylation of the HIF-alpha degradation domain node: id: hif_prolyl_hydroxylation label: Prolyl hydroxylation of HIF-alpha by PHD1-3 (EGLN2/EGLN1/EGLN3) module_type: REACTION description: >- Fe(II)- and 2-oxoglutarate-dependent dioxygenases hydroxylate two prolines in the oxygen-dependent degradation domain of HIF-alpha (Pro402 and Pro564 of HIF-1alpha), consuming O2 and 2-oxoglutarate and releasing succinate and CO2. Because O2 is a cosubstrate with a Km near the physiological range, the rate of this reaction is the cell's oxygen reading. The three paralogues catalyse the same chemistry and differ in abundance, localisation and site preference; PHD2 (EGLN1) sets the normoxic steady-state level of HIF-1alpha. concepts: - preferred_term: peptidyl-proline hydroxylation to 4-hydroxy-L-proline term: id: GO:0018401 label: peptidyl-proline hydroxylation to 4-hydroxy-L-proline evidence: - source_id: PMID:11292861 statement: >- Hydroxylation of HIF-1alpha Pro564 by a prolyl hydroxylase regulates the pVHL interaction. supporting_text: Here we show that the interaction between human pVHL and a specific domain of the HIF-1alpha subunit is regulated through hydroxylation of a proline residue (HIF-1alpha P564) by an enzyme we have termed HIF-alpha prolyl-hydroxylase (HIF-PH). - source_id: PMID:11598268 statement: >- The hydroxylases form a conserved family (the EGLN/PHD proteins) that is an essential component of the oxygen-sensing pathway. supporting_text: We identified a conserved family of HIF prolyl hydoxylase (HPH) enzymes that appear to be responsible for this posttranslational modification. - source_id: RHEA:48400 statement: >- L-prolyl-[hypoxia-inducible factor alpha subunit] + 2-oxoglutarate + O2 = trans-4-hydroxy-L-prolyl-[hypoxia-inducible factor alpha subunit] + succinate + CO2; the Rhea xref of GO:0160082 and EC 1.14.11.29. variant_sets: - id: phd_paralog label: HIF prolyl hydroxylase paralogue axis: enzyme paralogue (PHD1/PHD2/PHD3, differing in abundance, localisation and site preference) selection: ONE_OR_MORE variants: - id: egln1_phd2_variant label: PHD2 (EGLN1) — the dominant normoxic sensor module_type: REACTION annotons: - id: egln1_hif_prolyl_hydroxylase label: EGLN1 (PHD2) hypoxia-inducible factor-proline dioxygenase activity participant: selector_type: GENE_PRODUCT gene_product: preferred_term: EGLN1 (PHD2) term: id: UniProtKB:Q9GZT9 label: Egl nine homolog 1 function: preferred_term: hypoxia-inducible factor-proline dioxygenase activity term: id: GO:0160082 label: hypoxia-inducible factor-proline dioxygenase activity substrates: - preferred_term: L-prolyl-[HIF alpha subunit] term: id: CHEBI:50342 label: L-proline residue - preferred_term: 2-oxoglutarate term: id: CHEBI:16810 label: 2-oxoglutarate(2-) - preferred_term: O2 term: id: CHEBI:15379 label: dioxygen products: - preferred_term: trans-4-hydroxy-L-prolyl-[HIF alpha subunit] term: id: CHEBI:61965 label: trans-4-hydroxy-L-proline residue - preferred_term: succinate term: id: CHEBI:30031 label: succinate(2-) - preferred_term: CO2 term: id: CHEBI:16526 label: carbon dioxide targets: - preferred_term: HIF-1alpha term: id: UniProtKB:Q16665 label: Hypoxia-inducible factor 1-alpha - preferred_term: HIF-2alpha (EPAS1) term: id: UniProtKB:Q99814 label: Endothelial PAS domain-containing protein 1 processes: - preferred_term: negative regulation of hypoxia-inducible factor-1alpha signaling pathway term: id: GO:1902072 label: negative regulation of hypoxia-inducible factor-1alpha signaling pathway locations: - preferred_term: cytosol term: id: GO:0005829 label: cytosol role_description: >- The non-redundant oxygen sensor in cultured cells: silencing PHD2 alone stabilises and activates HIF-1alpha in normoxia, while silencing PHD1 or PHD3 does not. Its core function in the completed EGLN1 gene review is this activity, with GO:1902072 as the process. evidence: - source_id: PMID:12912907 statement: PHD2 knockdown alone is sufficient to stabilise HIF-1alpha in normoxia. supporting_text: Here we show that specific 'silencing' of PHD2 with short interfering RNAs is sufficient to stabilize and activate HIF-1alpha in normoxia in all the human cells investigated. - source_id: PMID:12912907 statement: PHD1 and PHD3 knockdown has no such effect, so the paralogues are not interchangeable. supporting_text: "'Silencing' of PHD1 and PHD3 has no effect on the stability of HIF-1alpha either in normoxia or upon re-oxygenation of cells briefly exposed to hypoxia." - id: egln2_phd1_variant label: PHD1 (EGLN2) module_type: REACTION annotons: - id: egln2_hif_prolyl_hydroxylase label: EGLN2 (PHD1) hypoxia-inducible factor-proline dioxygenase activity participant: selector_type: GENE_PRODUCT gene_product: preferred_term: EGLN2 (PHD1) term: id: UniProtKB:Q96KS0 label: Prolyl hydroxylase EGLN2 function: preferred_term: hypoxia-inducible factor-proline dioxygenase activity term: id: GO:0160082 label: hypoxia-inducible factor-proline dioxygenase activity targets: - preferred_term: HIF-1alpha term: id: UniProtKB:Q16665 label: Hypoxia-inducible factor 1-alpha locations: - preferred_term: nucleus term: id: GO:0005634 label: nucleus role_description: >- Predominantly nuclear paralogue, oestrogen-inducible, with a preference for the N-terminal degradation site. Dispensable for the normoxic set-point in cultured cells. evidence: - source_id: PMID:11598268 statement: >- Forced expression of an HPH family member attenuates normoxic HIF-1alpha accumulation, the assay that defines the family's activity. supporting_text: In cultured mammalian cells, inappropriate accumulation of HIF caused by forced expression of the HIF-1alpha subunit under normoxic conditions was attenuated by coexpression of HPH. - id: egln3_phd3_variant label: PHD3 (EGLN3) module_type: REACTION annotons: - id: egln3_hif_prolyl_hydroxylase label: EGLN3 (PHD3) hypoxia-inducible factor-proline dioxygenase activity participant: selector_type: GENE_PRODUCT gene_product: preferred_term: EGLN3 (PHD3) term: id: UniProtKB:Q9H6Z9 label: Prolyl hydroxylase EGLN3 function: preferred_term: hypoxia-inducible factor-proline dioxygenase activity term: id: GO:0160082 label: hypoxia-inducible factor-proline dioxygenase activity targets: - preferred_term: HIF-1alpha term: id: UniProtKB:Q16665 label: Hypoxia-inducible factor 1-alpha - preferred_term: HIF-2alpha (EPAS1) term: id: UniProtKB:Q99814 label: Endothelial PAS domain-containing protein 1 locations: - preferred_term: cytosol term: id: GO:0005829 label: cytosol role_description: >- Strongly hypoxia-inducible paralogue with a preference for the C-terminal degradation site and a marked preference for HIF-2alpha; also hydroxylates non-HIF substrates, which is what the only EGLN3 GO-CAM (gomodel:680ad14200007201, GPX4) models. evidence: - source_id: PMID:11598268 statement: >- The family was defined by the ability of its members to oppose normoxic HIF accumulation, and loss of the fly orthologue derepresses a hypoxia-inducible gene. supporting_text: Suppression of HPH in cultured Drosophila melanogaster cells by RNA interference resulted in elevated expression of a hypoxia-inducible gene (LDH, encoding lactate dehydrogenase) under normoxic conditions. - order: 2 role: scaffolding that promotes hydroxylation (oxygen-independent) optional: true node: id: os9_hydroxylation_scaffold label: OS-9 scaffolding of HIF-1alpha with the prolyl hydroxylases module_type: REGULATORY_STEP description: >- OS-9 binds both HIF-1alpha and the prolyl hydroxylases and raises the efficiency of hydroxylation and hence of VHL capture. It does not sense oxygen itself; removing it raises HIF-1alpha levels and HIF target-gene expression in non-hypoxic cells. annotons: - id: os9_scaffold label: OS-9 scaffold for HIF-1alpha prolyl hydroxylation participant: selector_type: GENE_PRODUCT gene_product: preferred_term: OS9 term: id: UniProtKB:Q13438 label: Protein OS-9 function: preferred_term: HIF-1alpha prolyl hydroxylase scaffold (no GO molecular function term) description: >- No GO molecular function describes this role. OS9's only experimental MF is GO:0140032 glycosylation-dependent protein binding (IDA), from its ERAD lectin function, which is a different activity; no identifier is asserted rather than choosing a plausible-looking term. targets: - preferred_term: HIF-1alpha term: id: UniProtKB:Q16665 label: Hypoxia-inducible factor 1-alpha - preferred_term: EGLN1 (PHD2) term: id: UniProtKB:Q9GZT9 label: Egl nine homolog 1 role_description: >- Multiprotein-complex component that couples the substrate to the sensor; its loss is a gain of HIF signalling, not a loss. evidence: - source_id: PMID:15721254 statement: OS-9 binds both HIF-1alpha and the HIF-1alpha prolyl hydroxylases. supporting_text: Here, we demonstrate that OS-9, the protein product of a widely expressed gene, interacts with both HIF-1alpha and HIF-1alpha prolyl hydroxylases. - source_id: PMID:15721254 statement: OS-9 gain of function promotes hydroxylation, VHL binding and degradation. supporting_text: OS-9 gain-of-function promotes HIF-1alpha hydroxylation, VHL binding, proteasomal degradation of HIF-1alpha, and inhibition of HIF-1-mediated transcription. - order: 3 role: recognition of hydroxyproline and ubiquitination by CRL2(VHL) node: id: crl2_vhl_degradation label: CRL2(VHL) recognition and ubiquitination of hydroxylated HIF-alpha module_type: REGULATORY_STEP description: >- pVHL binds the hydroxyprolyl degradation domain through a single hydroxyproline pocket in its beta-domain, with an affinity three orders of magnitude higher than for the unmodified peptide, while its alpha-domain (VHL-box) binds the Elongin B/C module. Elongin C bridges to the CUL2 scaffold, which carries the RING subunit RBX1; RBX1 recruits a ubiquitin-charged E2 and the alpha subunit is polyubiquitinated and degraded by the 26S proteasome. concepts: - preferred_term: VCB complex term: id: GO:0030891 label: VCB complex - preferred_term: Cul2-RING ubiquitin ligase complex term: id: GO:0031462 label: Cul2-RING ubiquitin ligase complex - preferred_term: proteasome-mediated ubiquitin-dependent protein catabolic process term: id: GO:0043161 label: proteasome-mediated ubiquitin-dependent protein catabolic process evidence: - source_id: PMID:11292862 statement: pVHL binding requires the hydroxylated proline, making hydroxylation the recognition mark. supporting_text: We found that human pVHL binds to a short HIF-derived peptide when a conserved proline residue at the core of this peptide is hydroxylated. - source_id: PMID:12050673 statement: >- The VCB (pVHL-elongin C-elongin B) structure shows one conserved hydroxyproline pocket that discriminates hydroxylated from unmodified prolyl residues. supporting_text: The crystal structure of a hydroxylated HIF-1 alpha peptide bound to VCB (pVHL, elongins C and B) and solution binding assays reveal a single, conserved hydroxyproline-binding pocket in pVHL. - source_id: PMID:12004076 statement: The hydroxyproline inserts into a gap in the pVHL hydrophobic core that is a tumour-mutation hotspot. supporting_text: The hydroxyproline inserts into a gap in the pVHL hydrophobic core, at a site that is a hotspot for tumorigenic mutations, with its 4-hydroxyl group recognized by buried serine and histidine residues. annotons: - id: vhl_substrate_adaptor label: VHL substrate receptor for hydroxylated HIF-alpha participant: selector_type: GENE_PRODUCT gene_product: preferred_term: VHL term: id: UniProtKB:P40337 label: von Hippel-Lindau disease tumor suppressor function: preferred_term: ubiquitin-like ligase-substrate adaptor activity term: id: GO:1990756 label: ubiquitin-like ligase-substrate adaptor activity targets: - preferred_term: hydroxyprolyl HIF-1alpha term: id: UniProtKB:Q16665 label: Hypoxia-inducible factor 1-alpha - preferred_term: hydroxyprolyl HIF-2alpha (EPAS1) term: id: UniProtKB:Q99814 label: Endothelial PAS domain-containing protein 1 processes: - preferred_term: proteasome-mediated ubiquitin-dependent protein catabolic process term: id: GO:0043161 label: proteasome-mediated ubiquitin-dependent protein catabolic process locations: - preferred_term: cytosol term: id: GO:0005829 label: cytosol role_description: >- The oxygen-dependent substrate receptor of the ligase: it reads the hydroxyproline mark with its beta-domain and delivers the alpha subunit to the cullin. Its loss, in VHL disease and clear-cell renal carcinoma, gives constitutive HIF activity. evidence: - source_id: PMID:10878807 statement: pVHL binds HIF directly through its beta-domain and targets it for ubiquitination, acting like an F-box substrate receptor. supporting_text: Here we show that pVHL, through its beta-domain, binds directly to hypoxia-inducible factor (HIF), thereby targeting HIF for ubiquitination in an alpha-domain-dependent manner. - source_id: PMID:10878807 statement: The function of pVHL in this complex is to recruit the substrate to the ubiquitination machinery. supporting_text: Furthermore, we provide the first direct evidence that pVHL has a function analogous to that of an F-box protein, namely, to recruit substrates to a ubiquitination machine. - id: eloc_adaptor label: Elongin C (ELOC) adaptor bridging VHL to CUL2 participant: selector_type: GENE_PRODUCT gene_product: preferred_term: ELOC term: id: UniProtKB:Q15369 label: Elongin-C function: preferred_term: protein-macromolecule adaptor activity term: id: GO:0030674 label: protein-macromolecule adaptor activity processes: - preferred_term: protein ubiquitination term: id: GO:0016567 label: protein ubiquitination locations: - preferred_term: cytosol term: id: GO:0005829 label: cytosol role_description: >- SKP1-like adaptor that binds the VHL-box and the CUL2 N-terminal domain; this adaptor activity is the core function of the completed ELOC gene review, which names the VHL-ELOB/ELOC-CUL2-RBX1 ligase explicitly. evidence: - source_id: PMID:9122164 statement: CUL2 association requires an intact trimeric pVHL-elongin B-C complex, so Elongin C is the bridge. supporting_text: The pVHL-Hs-CUL-2 association is strictly dependent on the integrity of the trimeric VBC complex. - id: elob_adaptor label: Elongin B (ELOB) ubiquitin-like partner of Elongin C participant: selector_type: GENE_PRODUCT gene_product: preferred_term: ELOB term: id: UniProtKB:Q15370 label: Elongin-B function: preferred_term: ubiquitin protein ligase binding term: id: GO:0031625 label: ubiquitin protein ligase binding processes: - preferred_term: protein ubiquitination term: id: GO:0016567 label: protein ubiquitination locations: - preferred_term: cytosol term: id: GO:0005829 label: cytosol role_description: >- Ubiquitin-like subunit that stabilises Elongin C and is part of the crystallised VCB complex; this ligase-binding activity is the core function of the completed ELOB gene review. evidence: - source_id: PMID:9122164 statement: pVHL forms a stable complex with elongin B and elongin C, the trimer that CUL2 then joins. supporting_text: The VHL protein (pVHL) has been shown to form a stable complex with elongin B and elongin C, two factors that stabilize and activate the transcription elongation factor elongin A. - id: cul2_scaffold label: CUL2 ubiquitin ligase complex scaffold participant: selector_type: GENE_PRODUCT gene_product: preferred_term: CUL2 term: id: UniProtKB:Q13617 label: Cullin-2 function: preferred_term: ubiquitin ligase complex scaffold activity term: id: GO:0160072 label: ubiquitin ligase complex scaffold activity processes: - preferred_term: proteasome-mediated ubiquitin-dependent protein catabolic process term: id: GO:0043161 label: proteasome-mediated ubiquitin-dependent protein catabolic process locations: - preferred_term: cytosol term: id: GO:0005829 label: cytosol role_description: >- Cullin scaffold that holds the VHL-Elongin B/C substrate-receptor module at one end and RBX1 at the other. evidence: - source_id: PMID:9122164 statement: CUL2 associates specifically with the pVHL-elongin B-C complex in vitro and in vivo. supporting_text: Here, Hs-CUL-2, a member of the recently identified multigene family, the cullins, is shown to specifically associate with the trimeric pVHL-elongin B-C (VBC) complex in vitro and in vivo. gocam_associations: - model: gomodel:63f809ec00000611 activity: gomodel:63f809ec00000611/63f809ec00000619 title: CUL2 ubiquitin ligase complex scaffold activity (GO:0160072) in a curated human CRL2 complex description: >- Grounds the CUL2 scaffold activity in a production GO-CAM. That model's substrate receptor is KLHDC10, not VHL, so it grounds the scaffold role and not the substrate choice. - id: rbx1_ring_ligase label: RBX1 RING ubiquitin ligase activity on HIF-alpha participant: selector_type: GENE_PRODUCT gene_product: preferred_term: RBX1 term: id: UniProtKB:P62877 label: E3 ubiquitin-protein ligase RBX1 function: preferred_term: ubiquitin protein ligase activity term: id: GO:0061630 label: ubiquitin protein ligase activity targets: - preferred_term: hydroxyprolyl HIF-1alpha term: id: UniProtKB:Q16665 label: Hypoxia-inducible factor 1-alpha processes: - preferred_term: proteasome-mediated ubiquitin-dependent protein catabolic process term: id: GO:0043161 label: proteasome-mediated ubiquitin-dependent protein catabolic process locations: - preferred_term: cytosol term: id: GO:0005829 label: cytosol role_description: >- RING subunit that positions the ubiquitin-charged E2 over the substrate; this activity, with proteasomal degradation as its process, is a core function of the completed RBX1 gene review. evidence: - source_id: PMID:10353251 statement: >- The alpha subunits are rapidly destroyed by the proteasome in oxygenated cells and the VHL complex is required for that destruction. supporting_text: The alpha subunits of HIF are rapidly degraded by the proteasome under normal conditions, but are stabilized by hypoxia. gocam_associations: - model: gomodel:63f809ec00000611 activity: gomodel:63f809ec00000611/63f809ec00000627 title: RBX1 ubiquitin protein ligase activity (GO:0061630) in a curated human CRL2 complex description: >- As for CUL2: grounds the RING activity within a CRL2 complex whose substrate receptor is KLHDC10 rather than VHL. - order: 4 role: stabilisation of the VHL-Elongin C interaction (oxygen-independent) optional: true node: id: sat2_vhl_stabilisation label: SSAT2 (SAT2) stabilisation of the VHL-Elongin C interaction module_type: REGULATORY_STEP description: >- SSAT2 makes multivalent contacts with HIF-1alpha, VHL and Elongin C and stabilises the VHL-Elongin C interaction, which is needed for the very rapid ubiquitination of the hydroxylated alpha subunit seen in oxygenated cells. Its polyamine acetyltransferase activity is not the activity at work here. annotons: - id: sat2_complex_stabiliser label: SSAT2 (SAT2) stabiliser of the VHL-Elongin C interaction participant: selector_type: GENE_PRODUCT gene_product: preferred_term: SAT2 (SSAT2) term: id: UniProtKB:Q96F10 label: Thialysine N-epsilon-acetyltransferase function: preferred_term: VHL-Elongin C interaction stabiliser (no GO molecular function term) description: >- No GO molecular function describes this role; SAT2's annotated activity is GO:0004145 diamine N-acetyltransferase activity, which is a different activity (and itself carries a NOT|enables IDA row in human GOA). No identifier is asserted. targets: - preferred_term: VHL term: id: UniProtKB:P40337 label: von Hippel-Lindau disease tumor suppressor - preferred_term: ELOC term: id: UniProtKB:Q15369 label: Elongin-C role_description: >- Accessory subunit of the ubiquitination complex in oxygenated cells; acts on the oxygen-dependent route, in contrast to its paralogue SSAT1. evidence: - source_id: PMID:17558023 statement: SSAT2 binds HIF-1alpha, VHL and Elongin C and stabilises the VHL-Elongin C interaction. supporting_text: SSAT2 binds to HIF-1alpha, VHL, and Elongin C and promotes ubiquitination of hydroxylated HIF-1alpha by stabilizing the interaction of VHL and Elongin C. - source_id: PMID:17558023 statement: The multivalent contacts are proposed to make complex formation efficient enough for the observed degradation rate. supporting_text: Multivalent interactions by SSAT2 provide a mechanism to ensure efficient complex formation, which is necessary for the extremely rapid ubiquitination and degradation of HIF-1alpha that is observed in oxygenated cells. - order: 5 role: oxygen-dependent asparaginyl hydroxylation that silences the transactivation domain node: id: fih_asparaginyl_hydroxylation label: Asparaginyl hydroxylation of the HIF-alpha C-TAD by FIH (HIF1AN) module_type: REACTION description: >- FIH hydroxylates a conserved asparagine (Asn803 of HIF-1alpha, Asn851 of HIF-2alpha) in the C-terminal transactivation domain, using O2 and 2-oxoglutarate as cosubstrates like the prolyl hydroxylases. 3-Hydroxyasparagine prevents the C-TAD from binding the CH1 domain of p300/CBP, so the alpha subunit that escapes degradation is still transcriptionally silent. The two hydroxylations are independent, and the alpha subunit becomes fully active only when both fail. FIH is not part of a feedback loop: like the prolyl hydroxylases it is a constitutive enzyme whose rate is set by oxygen availability. concepts: - preferred_term: protein hydroxylation term: id: GO:0018126 label: protein hydroxylation evidence: - source_id: PMID:12080085 statement: FIH-1 is the asparaginyl hydroxylase, an Fe(II)-dependent enzyme using molecular O2. supporting_text: Like known hydroxylase enzymes, FIH-1 is an Fe(II)-dependent enzyme that uses molecular O(2) to modify its substrate. - source_id: RHEA:54260 statement: >- L-asparaginyl-[protein] + 2-oxoglutarate + O2 = (3S)-3-hydroxy-L-asparaginyl-[protein] + succinate + CO2; a Rhea xref of GO:0036140 and EC 1.14.11.30. annotons: - id: hif1an_asparaginyl_hydroxylase label: HIF1AN (FIH) [protein]-asparagine 3-dioxygenase activity participant: selector_type: GENE_PRODUCT gene_product: preferred_term: HIF1AN (FIH-1) term: id: UniProtKB:Q9NWT6 label: Hypoxia-inducible factor 1-alpha inhibitor function: preferred_term: '[protein]-asparagine 3-dioxygenase activity' term: id: GO:0036140 label: '[protein]-asparagine 3-dioxygenase activity' substrates: - preferred_term: L-asparaginyl-[HIF alpha C-TAD] term: id: CHEBI:50347 label: L-asparagine residue - preferred_term: 2-oxoglutarate term: id: CHEBI:16810 label: 2-oxoglutarate(2-) - preferred_term: O2 term: id: CHEBI:15379 label: dioxygen products: - preferred_term: (3S)-3-hydroxy-L-asparaginyl-[HIF alpha C-TAD] term: id: CHEBI:138107 label: (3S)-3-hydroxy-L-asparagine residue - preferred_term: succinate term: id: CHEBI:30031 label: succinate(2-) - preferred_term: CO2 term: id: CHEBI:16526 label: carbon dioxide targets: - preferred_term: HIF-1alpha C-terminal transactivation domain term: id: UniProtKB:Q16665 label: Hypoxia-inducible factor 1-alpha - preferred_term: HIF-2alpha (EPAS1) C-terminal transactivation domain term: id: UniProtKB:Q99814 label: Endothelial PAS domain-containing protein 1 processes: - preferred_term: negative regulation of hypoxia-inducible factor-1alpha signaling pathway term: id: GO:1902072 label: negative regulation of hypoxia-inducible factor-1alpha signaling pathway locations: - preferred_term: nucleus term: id: GO:0005634 label: nucleus role_description: >- The transactivation-domain oxygen sensor. Unlike the prolyl hydroxylases it does not change the alpha subunit's abundance, only its ability to recruit coactivators; it also carries a transcription corepressor activity annotation (GO:0003714, IDA) consistent with that role. evidence: - source_id: PMID:11823643 statement: Hypoxic induction of the C-TAD works by abolishing hydroxylation of a conserved asparagine in it. supporting_text: We now show that hypoxic induction of the COOH-terminal transactivation domain (CAD) of HIF occurs through abrogation of hydroxylation of a conserved asparagine in the CAD. - source_id: PMID:11823643 statement: Blocking the dioxygenase lets the C-TAD bind p300, and an Asn-to-Ala substitution makes the interaction constitutive. supporting_text: Inhibitors of Fe(II)- and 2-oxoglutarate-dependent dioxygenases prevented hydroxylation of the Asn, thus allowing the CAD to interact with the p300 transcription coactivator. - order: 6 role: HIF-alpha/ARNT dimerisation and hypoxia response element binding node: id: hif_dimerisation label: HIF-alpha/ARNT heterodimer binding hypoxia response elements module_type: REGULATORY_STEP description: >- When hydroxylation fails, the alpha subunit escapes the ligase, accumulates and enters the nucleus, where its bHLH and PAS domains pair with those of the constitutive beta subunit. The heterodimer binds the hypoxia response element (RCGTG core) of target genes; neither subunit binds productively alone, so sequence-specific DNA binding is a property of the dimer. The beta subunit is shared with other bHLH-PAS factors, including the aryl hydrocarbon receptor, and is not oxygen-regulated. concepts: - preferred_term: RNA polymerase II transcription regulator complex term: id: GO:0090575 label: RNA polymerase II transcription regulator complex - preferred_term: positive regulation of transcription by RNA polymerase II term: id: GO:0045944 label: positive regulation of transcription by RNA polymerase II evidence: - source_id: PMID:7539918 statement: Both subunits are bHLH-PAS proteins, and the alpha subunit is the oxygen-regulated one. supporting_text: We show that both HIF-1 subunits are basic-helix-loop-helix proteins containing a PAS domain, defined by its presence in the Drosophila Per and Sim proteins and in the mammalian ARNT and AHR proteins. variant_sets: - id: hif_alpha_subunit label: HIF alpha subunit axis: oxygen-regulated alpha subunit paralogue (expression pattern and target repertoire) selection: ONE_OR_MORE variants: - id: hif1a_variant label: HIF-1alpha (HIF1A) module_type: REGULATORY_STEP annotons: - id: hif1a_transcription_factor label: HIF1A DNA-binding transcription factor activity participant: selector_type: GENE_PRODUCT gene_product: preferred_term: HIF1A (HIF-1alpha) term: id: UniProtKB:Q16665 label: Hypoxia-inducible factor 1-alpha function: preferred_term: DNA-binding transcription factor activity, RNA polymerase II-specific term: id: GO:0000981 label: DNA-binding transcription factor activity, RNA polymerase II-specific processes: - preferred_term: cellular response to hypoxia term: id: GO:0071456 label: cellular response to hypoxia locations: - preferred_term: nucleus term: id: GO:0005634 label: nucleus role_description: >- The broadly expressed alpha subunit and the one the pathway is named for; it carries the two hydroxylated prolines and the hydroxylated asparagine, so it is both the sensor's substrate and the dimer's activator. evidence: - source_id: PMID:7539918 statement: HIF-1 alpha and beta protein levels track oxygen tension, consistent with HIF-1 mediating the hypoxic transcriptional response. supporting_text: HIF-1 alpha and HIF-1 beta (ARNT) RNA and protein levels were induced in cells exposed to 1% O2 and decayed rapidly upon return of the cells to 20% O2, consistent with the role of HIF-1 as a mediator of transcriptional responses to hypoxia. gocam_associations: - model: gomodel:689e7a5d00004061 activity: gomodel:689e7a5d00004061/689e7a5d00004268 title: HIF1A DNA-binding transcription factor activity, RNA polymerase II-specific (GO:0000981), part of GO:0071456 cellular response to hypoxia - model: gomodel:689e7a5d00004412 activity: gomodel:689e7a5d00004412/689e7a5d00004413 title: HIF1A DNA-binding transcription factor activity, RNA polymerase II-specific (GO:0000981) in the lactate/COXFA4L2 model - id: epas1_variant label: HIF-2alpha (EPAS1) module_type: REGULATORY_STEP annotons: - id: epas1_transcription_factor label: EPAS1 (HIF-2alpha) DNA-binding transcription factor activity participant: selector_type: GENE_PRODUCT gene_product: preferred_term: EPAS1 (HIF-2alpha) term: id: UniProtKB:Q99814 label: Endothelial PAS domain-containing protein 1 function: preferred_term: DNA-binding transcription factor activity, RNA polymerase II-specific term: id: GO:0000981 label: DNA-binding transcription factor activity, RNA polymerase II-specific processes: - preferred_term: cellular response to hypoxia term: id: GO:0071456 label: cellular response to hypoxia locations: - preferred_term: nucleus term: id: GO:0005634 label: nucleus role_description: >- The restricted-expression alpha subunit (endothelium, kidney interstitium, carotid body, hepatocytes), regulated by the same hydroxylations but with a partly distinct target repertoire including EPO; gain-of-function mutations in its degradation domain cause erythrocytosis and paraganglioma. evidence: - source_id: PMID:9000051 statement: EPAS1 binds and activates a hypoxia response element and is stimulated by hypoxia. supporting_text: Activation by both HIF-1alpha and EPAS1 is stimulated by hypoxic conditions. - source_id: PMID:9000051 statement: EPAS1 heterodimerises with ARNT before activating transcription. supporting_text: EPAS1 forms a heterodimeric complex with the aryl hydrocarbon nuclear transporter prior to transcriptional activation of target genes. - id: hif3a_variant label: HIF-3alpha (HIF3A), including the inhibitory IPAS variant module_type: REGULATORY_STEP annotons: - id: hif3a_transcription_factor label: HIF3A DNA-binding transcription factor activity and IPAS dominant-negative role participant: selector_type: GENE_PRODUCT gene_product: preferred_term: HIF3A (HIF-3alpha / IPAS) term: id: UniProtKB:Q9Y2N7 label: Hypoxia-inducible factor 3-alpha function: preferred_term: DNA-binding transcription factor activity, RNA polymerase II-specific term: id: GO:0000981 label: DNA-binding transcription factor activity, RNA polymerase II-specific locations: - preferred_term: nucleus term: id: GO:0005634 label: nucleus role_description: >- The least understood alpha locus, and the only one whose products act in both directions. Full-length HIF-3alpha dimerises with ARNT and binds hypoxia response elements (GO:0000981 and GO:0000978 by IDA) but lacks a C-terminal transactivation domain, so it is a weak activator; the short splice variant IPAS has no transactivation function and acts as a dominant-negative on HIF-mediated gene expression. Modelled as one annoton with both readings rather than as a separate module, because the isoform repertoire is not resolved. evidence: - source_id: PMID:11734856 statement: The IPAS splice variant of this locus is a dominant-negative regulator of HIF-driven transcription. supporting_text: IPAS contains no endogenous transactivation function but demonstrates dominant negative regulation of HIF-mediated control of gene expression. - source_id: PMID:11734856 statement: Ectopic IPAS selectively impairs hypoxic induction of HIF target genes such as VEGF. supporting_text: Ectopic expression of IPAS in hepatoma cells selectively impairs induction of genes involved in adaptation to a hypoxic environment, notably the vascular endothelial growth factor (VEGF) gene, and results in retarded tumour growth and tumour vascular density in vivo. - id: hif_beta_subunit label: HIF beta subunit (ARNT family) axis: constitutive bHLH-PAS partner paralogue (tissue distribution) selection: ONE_OR_MORE variants: - id: arnt_variant label: ARNT (HIF-1beta) module_type: REGULATORY_STEP annotons: - id: arnt_heterodimer label: ARNT protein heterodimerization activity with HIF-alpha participant: selector_type: GENE_PRODUCT gene_product: preferred_term: ARNT (HIF-1beta) term: id: UniProtKB:P27540 label: Aryl hydrocarbon receptor nuclear translocator function: preferred_term: protein heterodimerization activity term: id: GO:0046982 label: protein heterodimerization activity targets: - preferred_term: HIF-1alpha term: id: UniProtKB:Q16665 label: Hypoxia-inducible factor 1-alpha - preferred_term: HIF-2alpha (EPAS1) term: id: UniProtKB:Q99814 label: Endothelial PAS domain-containing protein 1 processes: - preferred_term: response to hypoxia term: id: GO:0001666 label: response to hypoxia locations: - preferred_term: nucleus term: id: GO:0005634 label: nucleus role_description: >- The constitutive, non-oxygen-regulated partner. Its heterodimerization activity is a core function of the completed ARNT review, which also keeps the hypoxia-response annotations; the same subunit serves the aryl hydrocarbon receptor, so ARNT is not specific to this module. evidence: - source_id: PMID:7539918 statement: The beta subunit is an ARNT gene product that can dimerise with HIF-1alpha or with AHR. supporting_text: HIF-1 beta is a series of ARNT gene products, which can thus heterodimerize with either HIF-1 alpha or AHR. - id: arnt2_variant label: ARNT2 module_type: REGULATORY_STEP annotons: - id: arnt2_heterodimer label: ARNT2 protein heterodimerization activity participant: selector_type: GENE_PRODUCT gene_product: preferred_term: ARNT2 term: id: UniProtKB:Q9HBZ2 label: Aryl hydrocarbon receptor nuclear translocator 2 function: preferred_term: protein heterodimerization activity term: id: GO:0046982 label: protein heterodimerization activity targets: - preferred_term: HIF-1alpha term: id: UniProtKB:Q16665 label: Hypoxia-inducible factor 1-alpha locations: - preferred_term: nucleus term: id: GO:0005634 label: nucleus role_description: >- Second beta subunit, expressed mainly in brain and kidney, with overlapping but not identical partner specificity. Modelled as an alternative beta variant on the strength of its own experimental heterodimerization and HRE-binding annotations (GO:0046982 IDA, GO:0000981 IDA, GO:1990837 IDA) and the RGD record's statement that a second beta subunit with possibly overlapping functions exists; how much of the hypoxic response in those tissues runs through ARNT2 rather than ARNT is a knowledge gap. - order: 7 role: coactivator recruitment and transactivation node: id: coactivator_recruitment label: Recruitment of p300 (EP300) and CBP (CREBBP) by the HIF-alpha C-TAD module_type: REGULATORY_STEP description: >- The unhydroxylated C-terminal transactivation domain of the alpha subunit binds the CH1/TAZ1 domain of the paralogous acetyltransferase coactivators p300 and CBP, which bridge to the RNA polymerase II machinery and acetylate chromatin at hypoxia response elements. This is the step the FIH arm blocks, and it is where the switch becomes transcription. concepts: - preferred_term: positive regulation of transcription by RNA polymerase II term: id: GO:0045944 label: positive regulation of transcription by RNA polymerase II annotons: - id: ep300_coactivator label: EP300 (p300) transcription coactivator activity for HIF participant: selector_type: GENE_PRODUCT gene_product: preferred_term: EP300 (p300) term: id: UniProtKB:Q09472 label: Histone acetyltransferase p300 function: preferred_term: transcription coactivator activity term: id: GO:0003713 label: transcription coactivator activity targets: - preferred_term: HIF-1alpha term: id: UniProtKB:Q16665 label: Hypoxia-inducible factor 1-alpha processes: - preferred_term: positive regulation of transcription by RNA polymerase II term: id: GO:0045944 label: positive regulation of transcription by RNA polymerase II locations: - preferred_term: nucleus term: id: GO:0005634 label: nucleus role_description: >- The coactivator originally cloned as a HIF-1alpha partner; its coactivator activity is a core function of the completed EP300 review, whose response-to-hypoxia annotations are kept as non-core, matching the treatment here of p300 as a shared coactivator recruited by this pathway rather than a dedicated component. evidence: - source_id: PMID:8917528 statement: Hypoxia produces a DNA-binding complex containing HIF-1alpha and p300/CBP. supporting_text: Hypoxic conditions lead to the formation of a DNA binding complex containing both HIF-1 alpha and p300/CBP. - source_id: PMID:8917528 statement: >- p300 is limiting for hypoxic transcription; ectopic p300 enhances it and E1A sequestration inhibits it. supporting_text: Hypoxia-induced transcription from the Epo promoter was specifically enhanced by ectopic p300 and inhibited by E1A binding to p300/CBP. - id: crebbp_coactivator label: CREBBP (CBP) transcription coactivator activity for HIF participant: selector_type: GENE_PRODUCT gene_product: preferred_term: CREBBP (CBP) term: id: UniProtKB:Q92793 label: CREB-binding protein function: preferred_term: transcription coactivator activity term: id: GO:0003713 label: transcription coactivator activity targets: - preferred_term: HIF-1alpha term: id: UniProtKB:Q16665 label: Hypoxia-inducible factor 1-alpha processes: - preferred_term: positive regulation of transcription by RNA polymerase II term: id: GO:0045944 label: positive regulation of transcription by RNA polymerase II locations: - preferred_term: nucleus term: id: GO:0005634 label: nucleus role_description: >- Paralogue of p300, largely redundant with it for HIF transactivation and assayed together with it in the original work; coactivator activity is a core function of the completed CREBBP review. evidence: - source_id: PMID:8917528 statement: p300 and CBP are homologous coactivators assayed together as p300/CBP in the hypoxic response. supporting_text: p300 and CBP are homologous transcription adapters targeted by the E1A oncoprotein. - order: 8 role: oxygen-independent HIF-1alpha turnover (RACK1 route) optional: true node: id: oxygen_independent_turnover label: RACK1/SSAT1-dependent, VHL-independent ubiquitination of HIF-1alpha module_type: REGULATORY_STEP description: >- A second route to the same Elongin B/C ligase does not read hydroxyproline at all. RACK1 competes with HSP90 for the PAS-A domain of HIF-1alpha and, when bound, recruits the Elongin C ubiquitin-ligase complex, so HIF-1alpha is degraded independently of oxygen, of the prolyl hydroxylases and of VHL. SSAT1 stabilises the HIF-1alpha-RACK1 interaction, mirroring what its paralogue SSAT2 does for VHL-Elongin C. Marked optional: this arm modulates the set-point but is not required for the oxygen switch, and much of the evidence comes from HSP90 inhibition. annotons: - id: rack1_eloc_adaptor label: RACK1 adaptor recruiting Elongin C to HIF-1alpha participant: selector_type: GENE_PRODUCT gene_product: preferred_term: RACK1 term: id: UniProtKB:P63244 label: Small ribosomal subunit protein RACK1 function: preferred_term: molecular adaptor activity term: id: GO:0060090 label: molecular adaptor activity targets: - preferred_term: HIF-1alpha term: id: UniProtKB:Q16665 label: Hypoxia-inducible factor 1-alpha - preferred_term: ELOC term: id: UniProtKB:Q15369 label: Elongin-C processes: - preferred_term: proteasome-mediated ubiquitin-dependent protein catabolic process term: id: GO:0043161 label: proteasome-mediated ubiquitin-dependent protein catabolic process locations: - preferred_term: cytosol term: id: GO:0005829 label: cytosol role_description: >- Functional analogue of VHL on this route: it occupies the substrate-receptor position without recognising a hydroxyproline, and its Elongin C-binding site resembles VHL's. The broad GO:0060090 molecular adaptor activity is the only term that fits; it is a core function of the completed RACK1 review, which already records HIF1A as a degradation client. RACK1's main cellular role is ribosomal, so this is a peripheral activity for the protein but a real part of this module. evidence: - source_id: PMID:17244529 statement: RACK1 binds Elongin C and promotes HIF-1alpha ubiquitination. supporting_text: RACK1 binds to Elongin-C and promotes ubiquitination of HIF-1alpha. - source_id: PMID:17244529 statement: >- The route is independent of oxygen, the prolyl hydroxylases and VHL, and works by competition with HSP90 for the PAS-A domain. supporting_text: Thus, RACK1 is an essential component of an O(2)/PHD/VHL-independent mechanism for regulating HIF-1alpha stability through competition with HSP90 and recruitment of the Elongin-C/B ubiquitin ligase complex. - id: sat1_rack1_stabiliser label: SSAT1 (SAT1) stabiliser of the HIF-1alpha-RACK1 interaction participant: selector_type: GENE_PRODUCT gene_product: preferred_term: SAT1 (SSAT1) term: id: UniProtKB:P21673 label: Diamine acetyltransferase 1 function: preferred_term: HIF-1alpha-RACK1 interaction stabiliser (no GO molecular function term) description: >- No GO molecular function describes this role; SAT1's annotated and reviewed core activity is GO:0004145 diamine N-acetyltransferase activity in polyamine catabolism, a different activity. No identifier is asserted. targets: - preferred_term: HIF-1alpha term: id: UniProtKB:Q16665 label: Hypoxia-inducible factor 1-alpha - preferred_term: RACK1 term: id: UniProtKB:P63244 label: Small ribosomal subunit protein RACK1 role_description: >- Paralogue-mirrored accessory role: SSAT1 serves the oxygen-independent route as SSAT2 serves the oxygen-dependent one. evidence: - source_id: PMID:17875644 statement: SSAT1 binds HIF-1alpha and promotes its degradation by stabilising the HIF-1alpha-RACK1 interaction. supporting_text: However, in contrast to SSAT2, SSAT1 acts by stabilizing the interaction of HIF-1alpha with RACK1. - source_id: PMID:17875644 statement: The two paralogues are complementary, one oxygen-independent and one oxygen-dependent. supporting_text: Thus, the paralogs SSAT1 and SSAT2 play complementary roles in promoting O(2)-independent and O(2)-dependent degradation of HIF-1alpha. connections: - source: hif_prolyl_hydroxylation target: crl2_vhl_degradation connection_type: PROVIDES_INPUT_FOR description: >- trans-4-Hydroxyproline in the oxygen-dependent degradation domain is the mark that pVHL reads; without it the ligase does not engage the alpha subunit. chaining_status: NOT_APPLICABLE chaining_note: >- The intermediate is a modified protein residue, not a CoA thioester, so the CoA continuity check does not apply. The continuity that matters here is trans-4-hydroxy-L-proline residue (CHEBI:61965), the product of RHEA:48400 and the ligand of the pVHL hydroxyproline pocket. - source: os9_hydroxylation_scaffold target: hif_prolyl_hydroxylation connection_type: POSITIVELY_REGULATES description: OS-9 brings the substrate and the hydroxylases together, raising hydroxylation efficiency. - source: sat2_vhl_stabilisation target: crl2_vhl_degradation connection_type: POSITIVELY_REGULATES description: SSAT2 stabilises the VHL-Elongin C interaction and so the assembled ligase. - source: crl2_vhl_degradation target: hif_dimerisation connection_type: NEGATIVELY_REGULATES description: >- Polyubiquitination and proteasomal destruction of the alpha subunit prevent dimer formation; this is the normoxic off-state of the pathway. - source: hif_dimerisation target: coactivator_recruitment connection_type: PROVIDES_INPUT_FOR description: >- The DNA-bound heterodimer presents the C-terminal transactivation domain that p300/CBP bind. - source: fih_asparaginyl_hydroxylation target: coactivator_recruitment connection_type: NEGATIVELY_REGULATES description: >- 3-Hydroxyasparagine in the C-TAD blocks the CH1 interaction, silencing the alpha subunit that escaped degradation. This is the second, independent oxygen-sensing arm. - source: oxygen_independent_turnover target: hif_dimerisation connection_type: NEGATIVELY_REGULATES description: >- RACK1/SSAT1-driven ubiquitination lowers HIF-1alpha levels without reading oxygen, setting the amount of alpha subunit available in either condition. knowledge_gaps: - gap_statement: >- What the HIF3A locus does physiologically is unresolved. Its products range from a full-length ARNT-dimerising factor without a C-terminal transactivation domain to the dominant-negative IPAS, the splice repertoire and its tissue distribution are incompletely mapped, and no target-gene programme has been established for the activating form. boundary: >- HIF3A is modelled as a third variant of the alpha subunit in hif_alpha_subunit, carrying both the weak-activator and the dominant-negative readings on one annoton, rather than as a separate inhibitory module. gap_kind: - BIOLOGY status: OPEN significance: >- Whether HIF-3alpha is mainly a weak activator, a decoy that titrates ARNT away from HIF-1alpha and HIF-2alpha, or both in different tissues decides whether the module's alpha variant set is three parallel activators or two activators plus a competitive inhibitor, which changes the predicted dose-response of the whole switch. resolution: >- Isoform-resolved knock-ins at the Hif3a locus (full-length versus IPAS-only versus null) with matched ChIP-seq and RNA-seq in a tissue that expresses both, plus quantification of ARNT occupancy by each alpha subunit. provenance: - reference_id: file:projects/RGD_PATHWAY_ONTOLOGY/pathways/PW_0000360.yaml supporting_text: "the 3alpha gene is not well characterized - a splice variant appears to exert inhibitory roles" - gap_statement: >- How much of the hypoxic transcriptional response runs through ARNT2 rather than ARNT is unknown. ARNT2 dimerises with HIF-1alpha and binds DNA in assays and is expressed in brain and kidney, but no study has measured the HIF target-gene programme in ARNT2-null tissue with ARNT intact. boundary: >- ARNT2 is modelled as an alternative beta-subunit variant with the same heterodimerization activity as ARNT, not as a tissue-restricted replacement for it. gap_kind: - BIOLOGY status: OPEN significance: >- If ARNT2 carries a substantial share of the response in brain or kidney, the beta variant set is a genuine either/or in those tissues rather than a redundancy, and ARNT-targeted perturbations would under-report the hypoxic response there. resolution: >- Conditional Arnt2 deletion with Arnt intact (and the reciprocal) in brain and kidney, scoring canonical HIF targets under graded hypoxia, with subunit-resolved ChIP. provenance: - reference_id: file:projects/RGD_PATHWAY_ONTOLOGY/pathways/PW_0000360.yaml supporting_text: "A second beta subunit Arnt2 has been identified with possibly overlapping functions." - gap_statement: >- The molecular activities of the four oxygen-independent regulators (OS-9, SSAT1, SSAT2 and RACK1 in this role) are described only as interactions. Whether any of them changes the catalytic parameters of the hydroxylases or only the local concentration of substrate and enzyme, whether the acetyltransferase activity of SSAT1/SSAT2 is needed at all for their effect on HIF-1alpha, and what their stoichiometry in the complexes is are all open. boundary: >- These four are modelled as regulatory parts whose annotons carry a preferred_term and evidence but, except for RACK1's broad molecular adaptor activity, no GO molecular function identifier. gap_kind: - BIOLOGY status: OPEN significance: >- Until the activity is defined, no GO molecular function can be asserted for these proteins in this pathway, and it cannot be said whether they are catalytic accessories, scaffolds or stoichiometric subunits of the ligase and sensor complexes. resolution: >- Reconstituted hydroxylation and ubiquitination kinetics with and without each regulator, using catalytically dead SSAT1/SSAT2 point mutants to separate the acetyltransferase activity from the scaffolding role, plus quantitative native mass spectrometry of the assembled complexes. - gap_statement: >- The division of labour between PHD1, PHD2 and PHD3 in vivo is only partly understood. Knockdown shows PHD2 sets the normoxic HIF-1alpha level in cultured cells, but the site-, substrate- and tissue-preferences that make PHD1 and PHD3 non-redundant in whole animals, and the Km values for O2 that would say which of them reports graded hypoxia at physiological tensions, are not established. boundary: >- The three paralogues are modelled as a ONE_OR_MORE variant set over the same reaction (GO:0160082, RHEA:48400), with their differences recorded in role descriptions rather than as separate steps. gap_kind: - BIOLOGY status: OPEN significance: >- Which paralogue is the physiological oxygen sensor at a given tension determines whether the module behaves as one graded sensor or as a set of sensors with different thresholds, and it is the basis on which PHD inhibitors used for renal anaemia are judged selective. resolution: >- Steady-state kinetics of all three enzymes against both degradation-domain peptides across the physiological O2 range, with tissue-specific single and compound knockouts scored for HIF-1alpha and HIF-2alpha stabilisation. provenance: - reference_id: file:projects/RGD_PATHWAY_ONTOLOGY/pathways/PW_0000360.yaml supporting_text: "Of the three modifying prolyl hydrolases, Egln1 known as Phd2 appears to be essential for the hypoxic response."