{"gene":"EGLN1","run_date":"2026-06-09T23:54:42","timeline":{"discoveries":[{"year":2004,"finding":"PHD2 (EGLN1) hydroxylates specific prolyl residues on both HIF-1α and HIF-2α subunits in an oxygen-dependent manner, targeting them for proteolysis. siRNA-based knockdown demonstrated that PHD2 is the primary contributor to HIF regulation under normoxic conditions, and that PHD2 shows specificity for different prolyl hydroxylation sites within each HIF-α subunit and some selectivity between HIF-1α and HIF-2α isoforms.","method":"siRNA knockdown, isoform-specific PHD suppression assays in multiple cell types","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean RNAi loss-of-function with specific HIF-α hydroxylation site readouts, replicated across multiple cell types and independently replicated by other labs","pmids":["15247232"],"is_preprint":false},{"year":2005,"finding":"The PHD2 gene is transcribed exclusively from a downstream CpG island promoter that contains a functional hypoxia-responsive element (HRE). HIF-1 binds this HRE as demonstrated by EMSA, and the promoter is induced by hypoxia in luciferase reporter assays, establishing PHD2 as a direct HIF target gene and part of a negative-feedback loop.","method":"Promoter luciferase reporter assays, EMSA, sequence analysis, RT-PCR","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — multiple orthogonal methods (EMSA, reporter assays, transcript analysis) in one study demonstrating direct HIF-1 binding and functional HRE","pmids":["15563275"],"is_preprint":false},{"year":2005,"finding":"EGLN1/PHD2 represses HIF-1α transcriptional activity in hypoxia independently of promoting its proteolysis. Ectopic EGLN1 expression inhibited HIF-1α transcriptional activity without altering its protein levels in VHL-deficient cells, indicating a discrete transcriptional repression activity separate from the hydroxylation/degradation mechanism.","method":"Ectopic overexpression in VHL-deficient cells, luciferase reporter assays for HIF-1α transcriptional activity, siRNA knockdown","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — single lab, gain- and loss-of-function both supporting the same conclusion but mechanism of transcriptional repression not fully defined","pmids":["16157596"],"is_preprint":false},{"year":2000,"finding":"SM-20 (EGLN1) protein localizes to mitochondria via a functional N-terminal mitochondrial targeting sequence (first 25 amino acids), and promotes caspase-3-dependent apoptosis in NGF-deprived neurons. An N-terminally truncated form loses mitochondrial restriction but retains the ability to induce cell death, demonstrating that mitochondrial localization is not required for pro-apoptotic activity.","method":"Immunofluorescence co-localization with mitochondrial markers, SM-20/DHFR fusion protein import assays, caspase inhibitor experiments, cytochrome c fractionation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — reconstitution with fusion proteins to define targeting sequence, multiple orthogonal localization methods (co-staining, fractionation), functional caspase inhibitor rescue in single study","pmids":["11060309"],"is_preprint":false},{"year":2007,"finding":"Conditional inactivation of PHD2 (Egln1) in mice is sufficient to activate a subset of HIF target genes including erythropoietin, causing striking polycythemia and premature mortality from dilated cardiomyopathy, demonstrating PHD2 is the primary HIF prolyl hydroxylase in vivo.","method":"Conditional gene knockout in mice (Cre-lox), phenotypic analysis, gene expression profiling","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — rigorous conditional KO in vivo with defined molecular and physiological readouts, replicated conceptually across multiple labs","pmids":["18096761"],"is_preprint":false},{"year":2009,"finding":"PHD2 haplodeficiency in endothelial cells normalizes tumor vasculature, improves tumor perfusion and oxygenation, and inhibits metastasis. The mechanism involves HIF-driven upregulation of soluble VEGFR-1 and VE-cadherin, redirecting endothelial tip cell specification toward a quiescent phalanx formation.","method":"PHD2(+/-) mouse tumor implantation model, gene expression analysis, HIF pathway epistasis","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic model with mechanistic HIF pathway epistasis, published in high-impact journal with comprehensive analysis","pmids":["19217150"],"is_preprint":false},{"year":2009,"finding":"PHD2 regulates tumor angiogenesis and bone marrow-derived cell (BMDC) mobilization through IL-8 and angiogenin in a HIF-independent but NF-κB-dependent manner. PHD2 levels are decreased in human cancers versus normal tissue.","method":"PHD2 knockdown/overexpression in cancer cells, cytokine assays (ELISA), NF-κB pathway analysis","journal":"Cancer cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple cytokine and pathway readouts in a single lab; HIF-independence established by parallel experiments","pmids":["19477431"],"is_preprint":false},{"year":2009,"finding":"FKBP38 interacts with PHD2 via a glutamate-rich N-terminal domain in FKBP38 and the MYND-type zinc finger domain in PHD2's N-terminus. This interaction regulates PHD2 protein stability through a ubiquitin-independent proteasomal pathway dependent on membrane-anchored FKBP38. PHD2 subcellular localization overlaps with FKBP38 at the endoplasmic reticulum and mitochondria, with an additional cytoplasmic fraction.","method":"Peptide array binding assays, fluorescence spectroscopy, FRET, biochemical fractionation, immunofluorescence, co-immunoprecipitation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — multiple orthogonal biochemical and cell biology methods (FRET, peptide arrays, fractionation, co-IP) in single study defining domain-level interaction and stability mechanism","pmids":["19546213"],"is_preprint":false},{"year":2009,"finding":"PHD2 nuclear export requires CRM1 and the N-terminal 100 amino acids of PHD2. PHD2 cycles between nucleus and cytoplasm, and its nuclear import is independent of classical importin α/β receptors (unlike PHD1 which has a classical NLS-dependent import).","method":"Subcellular fractionation, CRM1 inhibition (leptomycin B), importin knockdown/competition assays, live cell imaging","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple pharmacological and genetic approaches to dissect nuclear import/export mechanisms in single study","pmids":["19631610"],"is_preprint":false},{"year":2011,"finding":"PHD2 haplodeficiency in macrophages skews them toward a pro-arteriogenic M2-like phenotype, promoting collateral artery growth via increased release of arteriogenic factors and smooth muscle cell recruitment. The mechanism relies on activation of the canonical NF-κB pathway in PHD2-haplodeficient macrophages.","method":"Phd2(+/-) mice, hindlimb ischemia model, macrophage-specific Phd2 deletion, NF-κB pathway analysis, macrophage depletion and transfer experiments","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — cell-type-specific genetic deletion, epistasis with NF-κB pathway, replicated with both chronic and acute deletion models","pmids":["21983962"],"is_preprint":false},{"year":2013,"finding":"PHD2's N-terminal MYND-type zinc finger domain binds a conserved Pro-Xaa-Leu-Glu (PXLE) motif in the HSP90 co-chaperones p23 and FKBP38. p23 knockdown augments hypoxia-induced HIF-1α levels and HIF target gene expression, indicating that the PHD2:p23 interaction facilitates HIF-α hydroxylation by linking PHD2 to the HSP90 chaperone pathway.","method":"Co-immunoprecipitation, PXLE motif identification, p23 siRNA knockdown, HIF-1α protein level measurement","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP and loss-of-function with defined functional readout in single lab","pmids":["23413029"],"is_preprint":false},{"year":2014,"finding":"The Tibetan PHD2 haplotype (D4E/C127S) strikingly diminishes PHD2 interaction with the HSP90 co-chaperone p23 via its PXLE motif, impairing PHD2-mediated downregulation of the HIF pathway. Both substitutions are required for the defective p23 binding. HSP90 itself also contains a PXLE motif that binds PHD2, but this interaction is maintained with the Tibetan haplotype.","method":"Co-immunoprecipitation, in vitro binding assays, HIF pathway reporter assays with Tibetan vs. wild-type PHD2","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and functional assays with defined mutants, single lab","pmids":["24711448"],"is_preprint":false},{"year":2015,"finding":"PHD2 hydroxylates proline residues P2309 and P2316 in the actin cross-linker filamin A (FLNA) under normoxia, leading to VHL-mediated ubiquitination and proteasomal degradation of FLNA. In hypoxia, PHD2 inactivation stabilizes FLNA, causing immature filopodium-like dendritic protrusions and reduced synaptic density independently of HIF signaling. Silencing FLNA rescues the immature spine phenotype caused by PHD2 inhibition.","method":"PHD2 siRNA, chemical PHD inhibition, site-directed mutagenesis of FLNA proline residues, ubiquitination assays, dendritic spine imaging, electrophysiology","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — site-specific mutagenesis of substrate combined with multiple orthogonal cell biology readouts; novel non-HIF substrate identified with functional validation","pmids":["26972007"],"is_preprint":false},{"year":2016,"finding":"PHD2 is phosphorylated at serine 125 (S125) by the mTOR downstream kinase P70S6K, increasing its ability to degrade HIF-1α. Under hypoxia, mTOR blockade by REDD1 restrains P70S6K and permits PP2A (via its regulatory subunit B55α) to directly dephosphorylate PHD2 at S125, reducing PHD2 activity and boosting HIF-1α accumulation to promote autophagy-dependent cell survival in colorectal cancer cells.","method":"Kinase assays, phospho-specific antibodies, mTOR/PP2A inhibitors, B55α knockdown, in vivo xenograft models, patient tissue analysis","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — identified specific phosphorylation site, multiple pathway inhibitors, in vitro and in vivo validation, patient tissue correlation in single comprehensive study","pmids":["28199842"],"is_preprint":false},{"year":2016,"finding":"PHD2 undergoes disulfide bond-mediated homo-dimerization in response to oxidative stress (oxidizing agents and oncogenic H-RasV12 signaling), and this dimerization inactivates PHD2, stabilizing HIF-1α. Cysteine residues in the double-stranded β-helix fold at the catalytic site mediate the oxidative dimerization. This mechanism links oncogenic RAS signaling to HIF-1α activation and aerobic glycolysis.","method":"Non-reducing SDS-PAGE, mutagenesis of cysteine residues, co-IP for dimer detection, antioxidant rescue experiments, PHD2 knockdown controls","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — biochemical dimerization assay with mutagenesis and antioxidant rescue, single lab","pmids":["26740011"],"is_preprint":false},{"year":2016,"finding":"Combined deletion of Phd2 and Phd3 in mice dramatically decreases phospholamban (PLN) expression, causes sustained CaMKII activation, and sensitizes mice to chronic β-adrenergic stress-induced myocardial injury. Mechanistically, PHD2 and PHD3 interact with and hydroxylate thyroid hormone receptor-α (TR-α) at two proline residues; PHD inhibition increases TR-α interaction with the corepressor NCOR2, suppressing PLN transcription.","method":"Double Phd2/Phd3 conditional knockout, co-IP of TR-α with PHD2/PHD3, in vitro prolyl hydroxylation assay of TR-α, transcript analysis, cardiac physiology","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — novel substrate (TR-α) identified with in vitro hydroxylation assay plus co-IP, in vivo genetic confirmation with defined mechanistic pathway","pmids":["26075818"],"is_preprint":false},{"year":2016,"finding":"PHD2 deficiency in endothelial/hematopoietic cells (Egln1Tie2 mice) causes severe pulmonary arterial hypertension with obliterative vascular remodeling via HIF-2α. Genetic deletion of both Egln1 and Hif2a (but not Hif1a) normalized the PAH phenotype. PHD2-deficient endothelial cells promote smooth muscle cell proliferation partly through HIF-2α-activated CXCL12 expression; genetic Cxcl12 deletion attenuated PAH.","method":"Tissue-specific double conditional knockout (Egln1/Hif1a and Egln1/Hif2a), Cxcl12 genetic deletion, hemodynamic measurements, histology","journal":"Circulation","confidence":"High","confidence_rationale":"Tier 2 / Strong — rigorous genetic epistasis with double KO models, pathway placement (PHD2→HIF-2α→CXCL12→SMC proliferation) validated by independent genetic rescue","pmids":["27143681"],"is_preprint":false},{"year":2016,"finding":"PHD2 is a regulator of glycolytic reprogramming in macrophages. PHD2-deficient macrophages show increased anaerobic glycolysis with increased PDK1 protein levels and decreased pyruvate dehydrogenase activity. Inhibition of PDK1 or knockout of HIF-1α reversed the metabolic phenotype, placing PHD2 upstream of HIF-1α/PDK1 in macrophage metabolic control.","method":"PHD2 knockout bone marrow-derived macrophages and RAW cells, metabolic phenotyping (Seahorse), HIF-1α knockout rescue, PDK1 inhibition","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell-type-specific KO with metabolic phenotype, epistasis via HIF-1α and PDK1 rescue; single lab","pmids":["27795296"],"is_preprint":false},{"year":2016,"finding":"PHD2 inhibition in pulmonary artery smooth muscle cells (PASMC) induces HIF-1α and promotes proliferation; SMC-specific PHD2 knockout exacerbates hypoxia-induced pulmonary hypertension and vascular remodeling in mice. The 3'-UTR of PHD2 contains a functional miR-17/20a binding site, and miR-17/20a suppresses PHD2 to upregulate HIF-1α and drive proliferation.","method":"SMC-specific PHD2 knockout mice, miR-17/20 inhibitors/mimics, 3'-UTR reporter assays, pulmonary hypertension hemodynamics","journal":"Journal of the American Heart Association","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo KO with hemodynamic phenotype and 3'-UTR reporter validation; single lab","pmids":["27919930"],"is_preprint":false},{"year":2017,"finding":"PHD2 hydroxylates the PP2A regulatory subunit B55α at proline 319, triggering its degradation. Under glucose starvation, PHD2-mediated B55α hydroxylation and degradation promotes apoptosis. PHD2 silencing rescues B55α from degradation and prevents apoptosis, while B55α knockdown restores apoptosis in PHD2-deficient cells, identifying B55α as a PHD2 substrate linking PHD2 to cell death responses under nutrient stress.","method":"PHD2 overexpression/silencing, site-directed mutagenesis of B55α proline 319, co-IP, in vitro hydroxylation, xenograft models","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — novel substrate with site-directed mutagenesis and in vitro hydroxylation assay; in vivo validation; single lab","pmids":["28329677"],"is_preprint":false},{"year":2019,"finding":"ERK directly phosphorylates PHD2, and this phosphorylation prevents PHD2 from binding to HIF-1α, thereby inhibiting HIF-1α hydroxylation and increasing HIF-1α stability. TGFβ1 activates this ERK/PHD2 signaling axis in platinum-resistant ovarian cancer to promote platinum resistance by stabilizing HIF-1α.","method":"In vitro kinase assay (ERK phosphorylating PHD2), co-IP of PHD2/HIF-1α, HIF-1α stability assays, TGFβ1 pathway inhibitors, in vivo xenograft models","journal":"Clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro kinase assay plus co-IP and in vivo validation, single lab","pmids":["31285371"],"is_preprint":false},{"year":2020,"finding":"Cystathionine β-synthase (CBS) produces H2S which persulfidates PHD2 at Cys21 and Cys33 in the zinc finger motif, augmenting its prolyl hydroxylase activity. Depletion of H2S (by hypoxia or CBS inhibition) reduces PHD2 persulfidation and inhibits its activity, resulting in HIF-1α stabilization. Point mutations at these cysteines confirmed their functional importance.","method":"Biochemical persulfidation assays, CBS knockdown/inhibition, site-directed mutagenesis of Cys21/Cys33, in vitro PHD2 activity assays, zebrafish CBS depletion model, H2S rescue experiments","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 1 / Moderate — novel PTM with in vitro hydroxylase activity assay, site-directed mutagenesis identifying specific residues, and in vivo zebrafish rescue, multiple orthogonal methods","pmids":["32937467"],"is_preprint":false},{"year":2020,"finding":"Additional PHD2 mutations at or near Asp-4 or Cys-127 (beyond the Tibetan D4E/C127S haplotype) impair interaction with the HSP90 cochaperone p23 in vitro. Mice carrying the Tibetan Phd2 allele display augmented hypoxic ventilatory response, and this phenotype is also produced by a p23 mutation that abrogates the PHD2:p23 interaction, confirming the Tibetan allele is a loss-of-function allele acting through the p23/HSP90 pathway.","method":"In vitro p23 binding assays with mutant PHD2, Tibetan Phd2 knockin mice, p23 mutant mice, plethysmography (hypoxic ventilatory response), HIF pathway analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro binding plus in vivo genetic knockin with defined physiological readout and p23 mutant phenocopy; mechanistically rigorous","pmids":["32414920"],"is_preprint":false},{"year":2013,"finding":"PHD2 shows substrate preference for the CODD (C-terminal oxygen degradation domain) of HIF-1α by approximately 20-fold over NODD. Kinetic competition assays and amide H/D exchange reveal that electrostatics influence this selectivity, and that both substrates stabilize the β2β3 loop to a similar extent; differences in substrate selectivity arise primarily from differential binding affinities rather than differential loop closure.","method":"In vitro kinetic competition assays, varied ionic strength, amide H/D exchange monitored by mass spectrometry","journal":"Journal of inorganic biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic assays with kinetic measurements and structural HDX-MS in a single focused biochemical study","pmids":["23787140"],"is_preprint":false},{"year":2013,"finding":"Novel crystal structure of PHD2 enzyme complexed with a 2,8-diazaspiro[4.5]decan-1-one inhibitor reveals previously unobserved binding interactions: hydrogen bond with Arg322, π-cation interaction with Arg322, π-π stacking with Trp389 and His313, without the salt bridge to Arg383 seen in all prior complex structures.","method":"X-ray crystallography of PHD2-inhibitor complex, structure-activity relationship studies","journal":"Bioorganic & medicinal chemistry","confidence":"High","confidence_rationale":"Tier 1 / Weak — crystal structure is Tier 1, but single study and focused on inhibitor binding rather than mechanistic substrate work","pmids":["24055079"],"is_preprint":false},{"year":2016,"finding":"Endothelial PHD2 deficiency induces obliterative pulmonary vascular remodeling and PAH through suppression of caveolin-1 (Cav1), which augments nitrative stress via endothelial NOS activation. Genetic restoration of Cav1 in Egln1Tie2 mice normalized nitrative stress and reduced PAH, while NOS3 knockdown or superoxide dismutase mimetic also inhibited the vascular remodeling.","method":"Egln1Tie2 mouse model, Cav1 genetic rescue, endothelial NOS3 knockdown via CRISPR-Cas9 nanoparticle delivery, SOD mimetic treatment, hemodynamic measurements","journal":"The European respiratory journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple genetic and pharmacological interventions in vivo, single lab","pmids":["35798360"],"is_preprint":false},{"year":2016,"finding":"PHD2 deficiency in endothelial cells and carotid bodies increases ventilatory sensitivity to hypoxia and causes carotid body hyperplasia through HIF-2α but not HIF-1α. Inducible inactivation of HIF-2α impairs ventilatory acclimatization to chronic hypoxia, establishing the PHD2/HIF-2α enzyme-substrate couple as the key mediator of hypoxic ventilatory responses.","method":"Tamoxifen-inducible Phd2 knockout, HIF-1α and HIF-2α conditional knockout epistasis, plethysmography, BrdU proliferation assays in carotid bodies","journal":"The Journal of physiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis in inducible models establishing PHD2/HIF-2α axis; replicated across constitutive and inducible deletion lines","pmids":["26337139"],"is_preprint":false},{"year":2016,"finding":"Inhibition of Egln1 systemically or in skeletal muscles protects mice against myocardial ischemia-reperfusion injury. The protection is mediated by a secreted factor: Egln1 loss causes accumulation of circulating α-ketoglutarate (αKG), which drives hepatic production and secretion of kynurenic acid (KYNA), which is necessary and sufficient to mediate cardiac ischemic protection.","method":"Somatic Egln1 gene deletion, pharmacological inhibition (PHD inhibitor), parabiosis experiments, metabolomics (αKG measurement), KYNA supplementation and depletion experiments","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — rigorous genetic and pharmacological models, parabiosis to define humoral factor, metabolomics to identify secreted αKG→KYNA axis; multiple orthogonal approaches","pmids":["26919427"],"is_preprint":false},{"year":2022,"finding":"Lactate, at physiological concentrations, directly binds to the catalytic domain of PHD2 in competition with α-ketoglutarate, inhibiting PHD2 activity and stabilizing HIF-1α. Lactate-induced IL-1β production was abolished in PHD2-deficient macrophages, identifying PHD2 as a direct metabolic sensor of lactate that connects chronic inflammation to energy metabolism.","method":"Direct binding assay (lactate to PHD2 catalytic domain), competitive inhibition kinetics vs. α-ketoglutarate, PHD2-deficient macrophage rescue experiments, in vivo adipocyte-specific LDHA deletion mouse model","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct binding/competitive inhibition assay with purified PHD2, genetic rescue in PHD2-deficient cells, in vivo mouse model; multiple orthogonal methods in single study","pmids":["36064857"],"is_preprint":false},{"year":2022,"finding":"SET7, a lysine monomethylase, catalyzes EGLN1 methylation at lysine 297, repressing EGLN1 prolyl hydroxylase activity toward HIF-1α. A methylation mimic mutant of EGLN1 loses the capability to suppress hypoxia signaling, leading to enhanced cell proliferation and increased oxygen consumption rate.","method":"In vitro methylation assay (SET7 acting on EGLN1), site-directed mutagenesis of K297, PHD2 activity assays, HIF-1α hydroxylation measurement, cell proliferation and metabolic assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic methylation assay plus site-directed mutagenesis with defined functional readout (prolyl hydroxylase activity); single study with multiple orthogonal methods","pmids":["35452683"],"is_preprint":false},{"year":2018,"finding":"DAP5 (Death-associated protein 5) regulates HIF-1α abundance by enabling DAP5:eIF2β complex-dependent translation of PHD2 during hypoxia. DAP5 depletion caused a surge in HIF-1α levels due to reduced PHD2 translation, revealing a translational control mechanism that safeguards against excessive HIF-1α accumulation during hypoxia-associated protein synthesis repression.","method":"DAP5 siRNA knockdown, polysome profiling for PHD2 mRNA, co-IP of DAP5:eIF2β, HIF-1α protein measurement, PHD2 reporter constructs","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus translational assay and HIF-1α functional readout; single lab","pmids":["29530922"],"is_preprint":false},{"year":2023,"finding":"EglN1/PHD2 accumulates on mitochondria under hypoxia via its β2β3 loop substrate-binding region, where it interacts with and prolyl-hydroxylates AMPKα under normoxia, causing their rapid dissociation and release from mitochondria. Under hypoxia, the EglN1-AMPKα interaction is maintained, and a CaMKK2-EglN1-AMPKα complex forms on mitochondria, activating AMPKα phosphorylation to maintain metabolic homeostasis and support breast tumor growth.","method":"Mitochondrial proteomic profiling, co-IP of EglN1-AMPKα on mitochondria, in vitro prolyl hydroxylation assay of AMPKα, β2β3 loop deletion mutants, CaMKK2 co-IP, xenograft tumor growth assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — novel mitochondrial substrate (AMPKα) identified by proteomics, in vitro hydroxylation assay, structural mutant validation of β2β3 loop, complex characterization; single lab with multiple orthogonal methods","pmids":["37661833"],"is_preprint":false},{"year":2023,"finding":"Osteocyte PHD2 (Egln1) controls FGF23 production: conditional osteocyte-specific Phd2 deletion upregulates Fgf23 through HIF-1α-dependent mechanisms. PHD2-knockout osteocyte-like cells lose iron-mediated suppression of Fgf23 that cannot be compensated by PHD1 or PHD3, and in vivo CKD models show suppressed Phd1-3 consistent with FGF23 upregulation, linking the oxygen/iron-sensing PHD2 axis to phosphate homeostasis.","method":"Osteocyte-specific Phd2 conditional knockout, CRISPR Phd2-KO osteocyte cell lines, ATACseq/RNAseq, HIF1α blockade rescue, chronic kidney disease mouse model","journal":"Bone research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — osteocyte-specific KO with genomic and transcriptional analyses plus HIF1α rescue; single lab","pmids":["36650133"],"is_preprint":false},{"year":2003,"finding":"Induction of SM-20 (EGLN1) expression in PC12 cells leads to increased cytochrome c levels, accumulation of cytochrome c in the cytosol, and caspase-dependent cell death, establishing a mechanism by which SM-20 promotes neuronal apoptosis through the cytochrome c/caspase pathway.","method":"Doxycycline-inducible SM-20 PC12 cell line, subcellular fractionation of cytochrome c, caspase activity assays, caspase inhibitor (zVAD-FMK) rescue","journal":"Journal of neurochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — inducible expression system with subcellular fractionation and pharmacological rescue; single lab","pmids":["12675908"],"is_preprint":false},{"year":2015,"finding":"PHD2 inhibition in cancer-associated fibroblasts (CAFs) phenocopies the effects of chronic hypoxia: PHD2 loss leads to HIF-1α stabilization, reduced αSMA and periostin expression, decreased myosin II activity, loss of contractile force, and reduced ECM remodeling. This deactivates CAFs and impairs CAF-mediated cancer cell invasion and metastasis. These effects require HIF-1α, as simultaneous HIF-1α depletion prevents the PHD2 loss phenotype.","method":"PHD2 RNAi and chemical inhibition (DMOG) in CAFs, orthotopic breast cancer model, αSMA/periostin expression analysis, traction force microscopy, HIF-1α siRNA epistasis","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic and pharmacological PHD2 loss-of-function with HIF-1α epistasis and in vivo tumor model; single lab","pmids":["26323721"],"is_preprint":false},{"year":2013,"finding":"PHD2 repression in macrophages is induced by angiopoietin-1 (ANG1)-mediated signaling during femoral artery occlusion. ANG1-dependent PHD2 repression initiates a feed-forward loop mediated by induction of the ANG receptor TIE2 in macrophages. TIE2 induction is required for proarteriogenic macrophage functions enabling collateral vessel formation.","method":"ANG1 blockade (soluble trap), Phd2 gene silencing in macrophages, macrophage depletion/transfer, TIE2 gene silencing, hindlimb ischemia model","journal":"EMBO molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic silencing and depletion strategies with defined ANG1-PHD2-TIE2 pathway; single lab","pmids":["23616286"],"is_preprint":false},{"year":2020,"finding":"Endothelial PHD2 deletion protects against renal ischemia-reperfusion injury by suppressing proinflammatory gene expression and inflammatory cell recruitment in a manner dependent on HIF-1 but not HIF-2. In vitro experiments implicated a humoral factor in the anti-inflammatory effects of endothelial PHD2/HIF-1 signaling.","method":"Endothelial-specific Phd2 deletion, double KO with HIF-1 or HIF-2, inducible endothelial Phd2 deletion in adult mice, renal ischemia-reperfusion injury model, inflammatory cell quantification","journal":"Journal of the American Society of Nephrology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with HIF isoform epistasis plus inducible deletion; single lab","pmids":["31996410"],"is_preprint":false},{"year":2021,"finding":"The gain-of-function Tibetan PHD2 D4E;C127S variant suppresses hypoxia-induced inflammatory responses in monocytes: PHD2D4E;C127S monocytes show reduced secretion of IL-6 and IL-1β, impaired chemotaxis, and downmodulation of RELA, JUN, STAT1, ATF2 and CXCR4. U937 monocytic cells engineered to express PHD2D4E;C127S confirmed these findings at the protein level under hypoxia. αKG supplementation (augmenting PHD2 activity) also diminished inflammatory responses in vitro and reduced leukocyte infiltration in lungs of hypoxia-exposed mice.","method":"Flow cytometry immunophenotyping of homozygous Tibetan PHD2 variant carriers, nCounter gene expression analysis, engineered U937 cell lines expressing Tibetan variant vs. WT PHD2, in vivo hypoxia mouse model with αKG supplementation","journal":"EBioMedicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ex vivo Tibetan cohort plus engineered cell line validation and in vivo rescue; single lab","pmids":["34102396"],"is_preprint":false}],"current_model":"EGLN1/PHD2 is an iron(II)- and 2-oxoglutarate-dependent dioxygenase that functions as the primary cellular oxygen sensor by prolyl-hydroxylating HIF-1α and HIF-2α (preferring the CODD site ~20-fold over NODD), targeting them for VHL-mediated ubiquitination and proteasomal degradation; beyond HIF-α, it also hydroxylates non-HIF substrates including filamin A (regulating dendritic spines), thyroid hormone receptor-α (regulating phospholamban and cardiac contractility), B55α/PP2A (regulating cell survival under nutrient stress), and AMPKα (on mitochondria during hypoxia to support metabolic homeostasis); its activity is modulated by multiple post-translational mechanisms including S125 phosphorylation by mTOR/P70S6K (activating) and PP2A/B55α-mediated dephosphorylation (inhibiting), K297 methylation by SET7 (inhibiting), persulfidation at Cys21/Cys33 by CBS-derived H2S (activating), oxidative disulfide-mediated homodimerization (inhibiting), and competitive inhibition by lactate at the α-KG binding site; PHD2 interacts with the HSP90 cochaperone p23 and FKBP38 via its N-terminal MYND zinc finger domain to link to the chaperone pathway, and cycles between cytoplasm and nucleus via CRM1-dependent export; in vivo, PHD2 is the dominant regulator of erythropoiesis, vascular normalization, arteriogenesis, ventilatory hypoxic response (through HIF-2α), immune regulation, and multiple organ-specific processes, and the Tibetan D4E/C127S haplotype represents a loss-of-function allele that impairs p23 binding and blunts HIF pathway activity to facilitate high-altitude adaptation."},"narrative":{"mechanistic_narrative":"EGLN1/PHD2 is an iron(II)- and 2-oxoglutarate-dependent prolyl hydroxylase that serves as the principal cellular oxygen sensor, hydroxylating specific prolyl residues on HIF-1α and HIF-2α to target them for proteolysis; loss-of-function studies establish PHD2 as the dominant HIF prolyl hydroxylase under normoxia and in vivo [PMID:15247232, PMID:18096761]. It preferentially hydroxylates the HIF-1α CODD site ~20-fold over NODD, a selectivity governed primarily by differential substrate binding affinity [PMID:23787140]. Because PHD2 is itself transcribed from a hypoxia-responsive promoter directly bound by HIF-1, the enzyme operates within a negative-feedback loop that restrains the HIF response [PMID:15563275], and additional layers of control include DAP5/eIF2β-dependent translation of PHD2 mRNA during hypoxia [PMID:29530922]. PHD2 activity is tuned by a dense array of post-translational and metabolic inputs: activating S125 phosphorylation by the mTOR/P70S6K axis opposed by PP2A-B55α dephosphorylation [PMID:28199842], ERK phosphorylation that blocks HIF-1α binding [PMID:31285371], inhibitory K297 methylation by SET7 [PMID:35452683], activating persulfidation at Cys21/Cys33 by CBS-derived H2S [PMID:32937467], inactivating oxidative disulfide homodimerization [PMID:26740011], and direct competitive inhibition at the α-ketoglutarate site by lactate [PMID:36064857]. Beyond HIF-α, PHD2 hydroxylates non-HIF substrates that connect it to diverse cellular programs: filamin A to control dendritic spine maturation [PMID:26972007], thyroid hormone receptor-α to regulate phospholamban and cardiac stress tolerance [PMID:26075818], the PP2A subunit B55α to govern apoptosis under glucose starvation [PMID:28329677], and mitochondrial AMPKα within a CaMKK2-containing complex to maintain metabolic homeostasis in hypoxia [PMID:37661833]. The enzyme links to the HSP90 chaperone pathway through its N-terminal MYND zinc finger, which binds a PXLE motif in the co-chaperones p23 and FKBP38 to facilitate HIF-α hydroxylation and regulate PHD2 stability [PMID:19546213, PMID:23413029], and it shuttles between cytoplasm and nucleus via CRM1-dependent export [PMID:19631610]. In vivo, PHD2 is the master regulator of erythropoiesis and cardiac homeostasis [PMID:18096761], tumor and ischemic vascular normalization and arteriogenesis [PMID:19217150, PMID:21983962], pulmonary vascular remodeling through HIF-2α [PMID:27143681], and the hypoxic ventilatory response via the PHD2/HIF-2α couple in the carotid body [PMID:26337139]. The Tibetan D4E/C127S haplotype is a loss-of-function allele that impairs p23 binding, blunting the HIF pathway and the hypoxic ventilatory/inflammatory response to facilitate high-altitude adaptation [PMID:32414920, PMID:34102396].","teleology":[{"year":2000,"claim":"Before its enzymatic identity was known, EGLN1 (SM-20) was found to localize to mitochondria and drive neuronal apoptosis, establishing an early functional readout that would later be reconciled with its dioxygenase role.","evidence":"fusion-protein import assays, co-localization, and caspase-inhibitor rescue in NGF-deprived neurons and PC12 cells","pmids":["11060309","12675908"],"confidence":"High","gaps":["did not identify the enzymatic activity or molecular substrate","relationship between pro-apoptotic activity and prolyl hydroxylase function unresolved"]},{"year":2004,"claim":"Defining EGLN1 as the dominant cellular HIF prolyl hydroxylase answered which enzyme sets normoxic HIF-α levels and revealed site- and isoform-selectivity.","evidence":"isoform-specific siRNA knockdown with HIF-α hydroxylation site readouts across multiple cell types","pmids":["15247232"],"confidence":"High","gaps":["kinetic basis of site selectivity not yet defined","did not address regulation of PHD2 itself"]},{"year":2005,"claim":"Identifying PHD2 as a direct HIF-1 target gene with a functional HRE explained how the oxygen-sensing system self-limits through negative feedback.","evidence":"promoter luciferase reporters, EMSA, and transcript analysis of the downstream CpG-island promoter","pmids":["15563275","16157596"],"confidence":"High","gaps":["mechanism of HIF-independent transcriptional repression by PHD2 not fully defined"]},{"year":2007,"claim":"Conditional mouse knockout established PHD2 as the primary HIF prolyl hydroxylase in vivo and tied it to erythropoiesis and cardiac homeostasis.","evidence":"Cre-lox conditional Egln1 inactivation with phenotypic and gene-expression profiling","pmids":["18096761"],"confidence":"High","gaps":["did not resolve which HIF isoform mediates each phenotype","tissue-specific contributions not dissected"]},{"year":2009,"claim":"Cell-type-specific PHD2 haplodeficiency revealed roles in endothelial vascular normalization and macrophage-driven arteriogenesis, including HIF-independent NF-κB-dependent outputs.","evidence":"Phd2(+/-) tumor and ischemia mouse models with HIF and NF-κB pathway epistasis and cytokine assays","pmids":["19217150","19477431"],"confidence":"High","gaps":["direct substrate underlying NF-κB/HIF-independent effects not identified"]},{"year":2009,"claim":"Mapping the N-terminal MYND zinc finger as a protein-interaction module connected PHD2 to chaperone-pathway partners and to non-canonical trafficking control.","evidence":"peptide arrays, FRET, co-IP, fractionation for FKBP38 binding, plus CRM1-inhibition and importin-competition for nucleocytoplasmic shuttling","pmids":["19546213","19631610"],"confidence":"High","gaps":["functional consequence of nuclear pool of PHD2 unresolved","FKBP38-dependent stability mechanism not linked to specific physiology"]},{"year":2013,"claim":"Biochemical and structural studies quantified CODD-over-NODD selectivity and defined inhibitor-binding chemistry of the catalytic site.","evidence":"kinetic competition assays, HDX-MS, and X-ray crystallography of PHD2-inhibitor complexes","pmids":["23787140","24055079"],"confidence":"High","gaps":["structural basis for non-HIF substrate recognition not addressed"]},{"year":2013,"claim":"Defining the PXLE motif shared by p23 and FKBP38, and later by HSP90, mechanistically linked PHD2 to the HSP90 chaperone machinery as a determinant of HIF-α hydroxylation.","evidence":"co-IP, PXLE motif identification, and p23 knockdown with HIF-1α readouts; ANG1-TIE2 feed-forward loop in macrophages","pmids":["23413029","23616286"],"confidence":"Medium","gaps":["how chaperone binding mechanistically enhances catalysis not defined","single-lab characterization of the p23 dependency"]},{"year":2015,"claim":"Identifying filamin A and cancer-associated fibroblast deactivation extended PHD2 function to HIF-independent substrate hydroxylation and tumor-stroma mechanics.","evidence":"site-directed mutagenesis of FLNA prolines, ubiquitination and spine imaging; CAF traction-force microscopy with HIF-1α epistasis","pmids":["26972007","26323721"],"confidence":"High","gaps":["physiological contexts of FLNA hydroxylation beyond neurons not mapped"]},{"year":2016,"claim":"A wave of in vivo and biochemical studies expanded the PHD2 substrate/regulatory repertoire (TR-α, oxidative dimerization, S125 phosphorylation) and dissected organ-specific HIF isoform dependencies in pulmonary, ventilatory, ischemic, and metabolic settings.","evidence":"double conditional KOs (Phd2/Phd3, Egln1/Hif1a, Egln1/Hif2a), in vitro hydroxylation of TR-α, cysteine mutagenesis, phospho-specific assays, parabiosis and metabolomics, Seahorse metabolic profiling","pmids":["26075818","26740011","28199842","27143681","26337139","26919427","27795296","27919930"],"confidence":"High","gaps":["integration of competing post-translational inputs in a single cell type unresolved","relative contribution of each non-HIF substrate to organ phenotypes unclear"]},{"year":2018,"claim":"Discovery of DAP5/eIF2β-dependent translation of PHD2 mRNA added a translational safeguard limiting HIF-1α accumulation during hypoxic protein-synthesis repression.","evidence":"DAP5 knockdown, polysome profiling of PHD2 mRNA, and DAP5:eIF2β co-IP","pmids":["29530922"],"confidence":"Medium","gaps":["single-lab characterization","in vivo relevance of translational control not established"]},{"year":2020,"claim":"Persulfidation by CBS-derived H2S and genetic confirmation of the Tibetan allele as a p23-pathway loss-of-function variant clarified how PHD2 activity is metabolically and evolutionarily tuned for hypoxic adaptation.","evidence":"persulfidation assays with Cys21/Cys33 mutagenesis and zebrafish rescue; Tibetan Phd2 and p23-mutant knockin mice with plethysmography; endothelial Cav1/NOS rescue in renal/pulmonary injury models","pmids":["32937467","32414920","35798360","31996410"],"confidence":"High","gaps":["interplay between persulfidation and other cysteine-based redox regulation not resolved"]},{"year":2022,"claim":"Direct lactate competition at the αKG site and SET7-mediated K297 methylation revealed PHD2 as a direct metabolic and epigenetic sensor coupling energy state to HIF signaling and inflammation.","evidence":"direct binding/competition kinetics with purified PHD2, in vitro methylation assays with K297 mutants, and PHD2-deficient macrophage rescue; Tibetan-variant monocyte immunophenotyping","pmids":["36064857","35452683","34102396"],"confidence":"High","gaps":["quantitative hierarchy among lactate, methylation, and phosphorylation inputs unknown"]},{"year":2023,"claim":"Identification of mitochondrial AMPKα as a substrate within a CaMKK2-EglN1-AMPKα complex and osteocyte control of FGF23 placed PHD2 at the interface of organelle-localized metabolic signaling and systemic phosphate/iron homeostasis.","evidence":"mitochondrial proteomics, in vitro AMPKα hydroxylation, β2β3 loop deletion mutants and xenografts; osteocyte-specific Phd2 KO with ATAC/RNAseq and HIF-1α rescue","pmids":["37661833","36650133"],"confidence":"High","gaps":["structural basis of mitochondrial recruitment via β2β3 loop not solved","generality of mitochondrial AMPKα hydroxylation across tissues untested"]},{"year":null,"claim":"How the many competing post-translational, redox, and metabolic regulators of PHD2 are integrated in real time within a single cell, and how substrate choice between HIF-α and the growing set of non-HIF substrates is determined, remain open.","evidence":"","pmids":[],"confidence":"Medium","gaps":["no unified model reconciling phosphorylation, methylation, persulfidation, dimerization, and lactate inhibition","substrate-selection rules for non-HIF targets undefined","structural determinants of organelle-specific PHD2 activity unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016491","term_label":"oxidoreductase activity","supporting_discovery_ids":[0,23,12,15,19,31]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,12,15,19,31]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0,23]},{"term_id":"GO:0140299","term_label":"molecular sensor activity","supporting_discovery_ids":[28,21]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[3,7,31]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[7,8]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[8]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[7]}],"pathway":[{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[0,4,26]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[17,27,28,31]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[1,2]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[9,28,36,37]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[3,33,19]}],"complexes":["CaMKK2-EglN1-AMPKα mitochondrial complex"],"partners":["HIF1A","EPAS1","FKBP38","PTGES3","HSP90","FLNA","THRA","PPP2R2A"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9GZT9","full_name":"Egl nine homolog 1","aliases":["Hypoxia-inducible factor prolyl hydroxylase 2","HIF-PH2","HIF-prolyl hydroxylase 2","HPH-2","Prolyl hydroxylase domain-containing protein 2","PHD2","SM-20"],"length_aa":426,"mass_kda":46.0,"function":"Cellular oxygen sensor that catalyzes, under normoxic conditions, the post-translational formation of 4-hydroxyproline in hypoxia-inducible factor (HIF) alpha proteins. Hydroxylates a specific proline found in each of the oxygen-dependent degradation (ODD) domains (N-terminal, NODD, and C-terminal, CODD) of HIF1A. Also hydroxylates HIF2A. Has a preference for the CODD site for both HIF1A and HIF1B. Hydroxylated HIFs are then targeted for proteasomal degradation via the von Hippel-Lindau ubiquitination complex. Under hypoxic conditions, the hydroxylation reaction is attenuated allowing HIFs to escape degradation resulting in their translocation to the nucleus, heterodimerization with HIF1B, and increased expression of hypoxy-inducible genes. EGLN1 is the most important isozyme under normoxia and, through regulating the stability of HIF1, involved in various hypoxia-influenced processes such as angiogenesis in retinal and cardiac functionality. Target proteins are preferentially recognized via a LXXLAP motif","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9GZT9/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/EGLN1","classification":"Not Classified","n_dependent_lines":229,"n_total_lines":1208,"dependency_fraction":0.18956953642384106},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"PTGES3","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/EGLN1","total_profiled":1310},"omim":[{"mim_id":"611463","title":"SPERMIDINE/SPERMINE N1-ACETYLTRANSFERASE 2; SAT2","url":"https://www.omim.org/entry/611463"},{"mim_id":"611118","title":"NUCLEAR PRELAMIN A RECOGNITION FACTOR-LIKE; NARFL","url":"https://www.omim.org/entry/611118"},{"mim_id":"610417","title":"SCAN DOMAIN-CONTAINING 2; SCAND2","url":"https://www.omim.org/entry/610417"},{"mim_id":"609820","title":"ERYTHROCYTOSIS, FAMILIAL, 3; ECYT3","url":"https://www.omim.org/entry/609820"},{"mim_id":"609070","title":"HEMOGLOBIN, HIGH ALTITUDE ADAPTATION; HALAH","url":"https://www.omim.org/entry/609070"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"},{"location":"Vesicles","reliability":"Additional"}],"tissue_specificity":"Group enriched","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"skeletal muscle","ntpm":396.2},{"tissue":"tongue","ntpm":143.2}],"url":"https://www.proteinatlas.org/search/EGLN1"},"hgnc":{"alias_symbol":["SM-20","PHD2","ZMYND6","HIFPH2"],"prev_symbol":["C1orf12"]},"alphafold":{"accession":"Q9GZT9","domains":[{"cath_id":"-","chopping":"18-54","consensus_level":"high","plddt":88.0059,"start":18,"end":54},{"cath_id":"2.60.120.620","chopping":"189-404","consensus_level":"high","plddt":94.0385,"start":189,"end":404}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9GZT9","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9GZT9-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9GZT9-F1-predicted_aligned_error_v6.png","plddt_mean":71.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=EGLN1","jax_strain_url":"https://www.jax.org/strain/search?query=EGLN1"},"sequence":{"accession":"Q9GZT9","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9GZT9.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9GZT9/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9GZT9"}},"corpus_meta":[{"pmid":"15247232","id":"PMC_15247232","title":"Differential 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HIF-1 binds this HRE as demonstrated by EMSA, and the promoter is induced by hypoxia in luciferase reporter assays, establishing PHD2 as a direct HIF target gene and part of a negative-feedback loop.\",\n      \"method\": \"Promoter luciferase reporter assays, EMSA, sequence analysis, RT-PCR\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — multiple orthogonal methods (EMSA, reporter assays, transcript analysis) in one study demonstrating direct HIF-1 binding and functional HRE\",\n      \"pmids\": [\"15563275\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"EGLN1/PHD2 represses HIF-1α transcriptional activity in hypoxia independently of promoting its proteolysis. Ectopic EGLN1 expression inhibited HIF-1α transcriptional activity without altering its protein levels in VHL-deficient cells, indicating a discrete transcriptional repression activity separate from the hydroxylation/degradation mechanism.\",\n      \"method\": \"Ectopic overexpression in VHL-deficient cells, luciferase reporter assays for HIF-1α transcriptional activity, siRNA knockdown\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — single lab, gain- and loss-of-function both supporting the same conclusion but mechanism of transcriptional repression not fully defined\",\n      \"pmids\": [\"16157596\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"SM-20 (EGLN1) protein localizes to mitochondria via a functional N-terminal mitochondrial targeting sequence (first 25 amino acids), and promotes caspase-3-dependent apoptosis in NGF-deprived neurons. An N-terminally truncated form loses mitochondrial restriction but retains the ability to induce cell death, demonstrating that mitochondrial localization is not required for pro-apoptotic activity.\",\n      \"method\": \"Immunofluorescence co-localization with mitochondrial markers, SM-20/DHFR fusion protein import assays, caspase inhibitor experiments, cytochrome c fractionation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — reconstitution with fusion proteins to define targeting sequence, multiple orthogonal localization methods (co-staining, fractionation), functional caspase inhibitor rescue in single study\",\n      \"pmids\": [\"11060309\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Conditional inactivation of PHD2 (Egln1) in mice is sufficient to activate a subset of HIF target genes including erythropoietin, causing striking polycythemia and premature mortality from dilated cardiomyopathy, demonstrating PHD2 is the primary HIF prolyl hydroxylase in vivo.\",\n      \"method\": \"Conditional gene knockout in mice (Cre-lox), phenotypic analysis, gene expression profiling\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — rigorous conditional KO in vivo with defined molecular and physiological readouts, replicated conceptually across multiple labs\",\n      \"pmids\": [\"18096761\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"PHD2 haplodeficiency in endothelial cells normalizes tumor vasculature, improves tumor perfusion and oxygenation, and inhibits metastasis. The mechanism involves HIF-driven upregulation of soluble VEGFR-1 and VE-cadherin, redirecting endothelial tip cell specification toward a quiescent phalanx formation.\",\n      \"method\": \"PHD2(+/-) mouse tumor implantation model, gene expression analysis, HIF pathway epistasis\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic model with mechanistic HIF pathway epistasis, published in high-impact journal with comprehensive analysis\",\n      \"pmids\": [\"19217150\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"PHD2 regulates tumor angiogenesis and bone marrow-derived cell (BMDC) mobilization through IL-8 and angiogenin in a HIF-independent but NF-κB-dependent manner. PHD2 levels are decreased in human cancers versus normal tissue.\",\n      \"method\": \"PHD2 knockdown/overexpression in cancer cells, cytokine assays (ELISA), NF-κB pathway analysis\",\n      \"journal\": \"Cancer cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple cytokine and pathway readouts in a single lab; HIF-independence established by parallel experiments\",\n      \"pmids\": [\"19477431\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"FKBP38 interacts with PHD2 via a glutamate-rich N-terminal domain in FKBP38 and the MYND-type zinc finger domain in PHD2's N-terminus. This interaction regulates PHD2 protein stability through a ubiquitin-independent proteasomal pathway dependent on membrane-anchored FKBP38. PHD2 subcellular localization overlaps with FKBP38 at the endoplasmic reticulum and mitochondria, with an additional cytoplasmic fraction.\",\n      \"method\": \"Peptide array binding assays, fluorescence spectroscopy, FRET, biochemical fractionation, immunofluorescence, co-immunoprecipitation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — multiple orthogonal biochemical and cell biology methods (FRET, peptide arrays, fractionation, co-IP) in single study defining domain-level interaction and stability mechanism\",\n      \"pmids\": [\"19546213\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"PHD2 nuclear export requires CRM1 and the N-terminal 100 amino acids of PHD2. PHD2 cycles between nucleus and cytoplasm, and its nuclear import is independent of classical importin α/β receptors (unlike PHD1 which has a classical NLS-dependent import).\",\n      \"method\": \"Subcellular fractionation, CRM1 inhibition (leptomycin B), importin knockdown/competition assays, live cell imaging\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple pharmacological and genetic approaches to dissect nuclear import/export mechanisms in single study\",\n      \"pmids\": [\"19631610\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"PHD2 haplodeficiency in macrophages skews them toward a pro-arteriogenic M2-like phenotype, promoting collateral artery growth via increased release of arteriogenic factors and smooth muscle cell recruitment. The mechanism relies on activation of the canonical NF-κB pathway in PHD2-haplodeficient macrophages.\",\n      \"method\": \"Phd2(+/-) mice, hindlimb ischemia model, macrophage-specific Phd2 deletion, NF-κB pathway analysis, macrophage depletion and transfer experiments\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — cell-type-specific genetic deletion, epistasis with NF-κB pathway, replicated with both chronic and acute deletion models\",\n      \"pmids\": [\"21983962\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PHD2's N-terminal MYND-type zinc finger domain binds a conserved Pro-Xaa-Leu-Glu (PXLE) motif in the HSP90 co-chaperones p23 and FKBP38. p23 knockdown augments hypoxia-induced HIF-1α levels and HIF target gene expression, indicating that the PHD2:p23 interaction facilitates HIF-α hydroxylation by linking PHD2 to the HSP90 chaperone pathway.\",\n      \"method\": \"Co-immunoprecipitation, PXLE motif identification, p23 siRNA knockdown, HIF-1α protein level measurement\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP and loss-of-function with defined functional readout in single lab\",\n      \"pmids\": [\"23413029\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The Tibetan PHD2 haplotype (D4E/C127S) strikingly diminishes PHD2 interaction with the HSP90 co-chaperone p23 via its PXLE motif, impairing PHD2-mediated downregulation of the HIF pathway. Both substitutions are required for the defective p23 binding. HSP90 itself also contains a PXLE motif that binds PHD2, but this interaction is maintained with the Tibetan haplotype.\",\n      \"method\": \"Co-immunoprecipitation, in vitro binding assays, HIF pathway reporter assays with Tibetan vs. wild-type PHD2\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and functional assays with defined mutants, single lab\",\n      \"pmids\": [\"24711448\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"PHD2 hydroxylates proline residues P2309 and P2316 in the actin cross-linker filamin A (FLNA) under normoxia, leading to VHL-mediated ubiquitination and proteasomal degradation of FLNA. In hypoxia, PHD2 inactivation stabilizes FLNA, causing immature filopodium-like dendritic protrusions and reduced synaptic density independently of HIF signaling. Silencing FLNA rescues the immature spine phenotype caused by PHD2 inhibition.\",\n      \"method\": \"PHD2 siRNA, chemical PHD inhibition, site-directed mutagenesis of FLNA proline residues, ubiquitination assays, dendritic spine imaging, electrophysiology\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — site-specific mutagenesis of substrate combined with multiple orthogonal cell biology readouts; novel non-HIF substrate identified with functional validation\",\n      \"pmids\": [\"26972007\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PHD2 is phosphorylated at serine 125 (S125) by the mTOR downstream kinase P70S6K, increasing its ability to degrade HIF-1α. Under hypoxia, mTOR blockade by REDD1 restrains P70S6K and permits PP2A (via its regulatory subunit B55α) to directly dephosphorylate PHD2 at S125, reducing PHD2 activity and boosting HIF-1α accumulation to promote autophagy-dependent cell survival in colorectal cancer cells.\",\n      \"method\": \"Kinase assays, phospho-specific antibodies, mTOR/PP2A inhibitors, B55α knockdown, in vivo xenograft models, patient tissue analysis\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — identified specific phosphorylation site, multiple pathway inhibitors, in vitro and in vivo validation, patient tissue correlation in single comprehensive study\",\n      \"pmids\": [\"28199842\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PHD2 undergoes disulfide bond-mediated homo-dimerization in response to oxidative stress (oxidizing agents and oncogenic H-RasV12 signaling), and this dimerization inactivates PHD2, stabilizing HIF-1α. Cysteine residues in the double-stranded β-helix fold at the catalytic site mediate the oxidative dimerization. This mechanism links oncogenic RAS signaling to HIF-1α activation and aerobic glycolysis.\",\n      \"method\": \"Non-reducing SDS-PAGE, mutagenesis of cysteine residues, co-IP for dimer detection, antioxidant rescue experiments, PHD2 knockdown controls\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — biochemical dimerization assay with mutagenesis and antioxidant rescue, single lab\",\n      \"pmids\": [\"26740011\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Combined deletion of Phd2 and Phd3 in mice dramatically decreases phospholamban (PLN) expression, causes sustained CaMKII activation, and sensitizes mice to chronic β-adrenergic stress-induced myocardial injury. Mechanistically, PHD2 and PHD3 interact with and hydroxylate thyroid hormone receptor-α (TR-α) at two proline residues; PHD inhibition increases TR-α interaction with the corepressor NCOR2, suppressing PLN transcription.\",\n      \"method\": \"Double Phd2/Phd3 conditional knockout, co-IP of TR-α with PHD2/PHD3, in vitro prolyl hydroxylation assay of TR-α, transcript analysis, cardiac physiology\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — novel substrate (TR-α) identified with in vitro hydroxylation assay plus co-IP, in vivo genetic confirmation with defined mechanistic pathway\",\n      \"pmids\": [\"26075818\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PHD2 deficiency in endothelial/hematopoietic cells (Egln1Tie2 mice) causes severe pulmonary arterial hypertension with obliterative vascular remodeling via HIF-2α. Genetic deletion of both Egln1 and Hif2a (but not Hif1a) normalized the PAH phenotype. PHD2-deficient endothelial cells promote smooth muscle cell proliferation partly through HIF-2α-activated CXCL12 expression; genetic Cxcl12 deletion attenuated PAH.\",\n      \"method\": \"Tissue-specific double conditional knockout (Egln1/Hif1a and Egln1/Hif2a), Cxcl12 genetic deletion, hemodynamic measurements, histology\",\n      \"journal\": \"Circulation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — rigorous genetic epistasis with double KO models, pathway placement (PHD2→HIF-2α→CXCL12→SMC proliferation) validated by independent genetic rescue\",\n      \"pmids\": [\"27143681\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PHD2 is a regulator of glycolytic reprogramming in macrophages. PHD2-deficient macrophages show increased anaerobic glycolysis with increased PDK1 protein levels and decreased pyruvate dehydrogenase activity. Inhibition of PDK1 or knockout of HIF-1α reversed the metabolic phenotype, placing PHD2 upstream of HIF-1α/PDK1 in macrophage metabolic control.\",\n      \"method\": \"PHD2 knockout bone marrow-derived macrophages and RAW cells, metabolic phenotyping (Seahorse), HIF-1α knockout rescue, PDK1 inhibition\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell-type-specific KO with metabolic phenotype, epistasis via HIF-1α and PDK1 rescue; single lab\",\n      \"pmids\": [\"27795296\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PHD2 inhibition in pulmonary artery smooth muscle cells (PASMC) induces HIF-1α and promotes proliferation; SMC-specific PHD2 knockout exacerbates hypoxia-induced pulmonary hypertension and vascular remodeling in mice. The 3'-UTR of PHD2 contains a functional miR-17/20a binding site, and miR-17/20a suppresses PHD2 to upregulate HIF-1α and drive proliferation.\",\n      \"method\": \"SMC-specific PHD2 knockout mice, miR-17/20 inhibitors/mimics, 3'-UTR reporter assays, pulmonary hypertension hemodynamics\",\n      \"journal\": \"Journal of the American Heart Association\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo KO with hemodynamic phenotype and 3'-UTR reporter validation; single lab\",\n      \"pmids\": [\"27919930\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"PHD2 hydroxylates the PP2A regulatory subunit B55α at proline 319, triggering its degradation. Under glucose starvation, PHD2-mediated B55α hydroxylation and degradation promotes apoptosis. PHD2 silencing rescues B55α from degradation and prevents apoptosis, while B55α knockdown restores apoptosis in PHD2-deficient cells, identifying B55α as a PHD2 substrate linking PHD2 to cell death responses under nutrient stress.\",\n      \"method\": \"PHD2 overexpression/silencing, site-directed mutagenesis of B55α proline 319, co-IP, in vitro hydroxylation, xenograft models\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — novel substrate with site-directed mutagenesis and in vitro hydroxylation assay; in vivo validation; single lab\",\n      \"pmids\": [\"28329677\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"ERK directly phosphorylates PHD2, and this phosphorylation prevents PHD2 from binding to HIF-1α, thereby inhibiting HIF-1α hydroxylation and increasing HIF-1α stability. TGFβ1 activates this ERK/PHD2 signaling axis in platinum-resistant ovarian cancer to promote platinum resistance by stabilizing HIF-1α.\",\n      \"method\": \"In vitro kinase assay (ERK phosphorylating PHD2), co-IP of PHD2/HIF-1α, HIF-1α stability assays, TGFβ1 pathway inhibitors, in vivo xenograft models\",\n      \"journal\": \"Clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro kinase assay plus co-IP and in vivo validation, single lab\",\n      \"pmids\": [\"31285371\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Cystathionine β-synthase (CBS) produces H2S which persulfidates PHD2 at Cys21 and Cys33 in the zinc finger motif, augmenting its prolyl hydroxylase activity. Depletion of H2S (by hypoxia or CBS inhibition) reduces PHD2 persulfidation and inhibits its activity, resulting in HIF-1α stabilization. Point mutations at these cysteines confirmed their functional importance.\",\n      \"method\": \"Biochemical persulfidation assays, CBS knockdown/inhibition, site-directed mutagenesis of Cys21/Cys33, in vitro PHD2 activity assays, zebrafish CBS depletion model, H2S rescue experiments\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — novel PTM with in vitro hydroxylase activity assay, site-directed mutagenesis identifying specific residues, and in vivo zebrafish rescue, multiple orthogonal methods\",\n      \"pmids\": [\"32937467\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Additional PHD2 mutations at or near Asp-4 or Cys-127 (beyond the Tibetan D4E/C127S haplotype) impair interaction with the HSP90 cochaperone p23 in vitro. Mice carrying the Tibetan Phd2 allele display augmented hypoxic ventilatory response, and this phenotype is also produced by a p23 mutation that abrogates the PHD2:p23 interaction, confirming the Tibetan allele is a loss-of-function allele acting through the p23/HSP90 pathway.\",\n      \"method\": \"In vitro p23 binding assays with mutant PHD2, Tibetan Phd2 knockin mice, p23 mutant mice, plethysmography (hypoxic ventilatory response), HIF pathway analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro binding plus in vivo genetic knockin with defined physiological readout and p23 mutant phenocopy; mechanistically rigorous\",\n      \"pmids\": [\"32414920\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PHD2 shows substrate preference for the CODD (C-terminal oxygen degradation domain) of HIF-1α by approximately 20-fold over NODD. Kinetic competition assays and amide H/D exchange reveal that electrostatics influence this selectivity, and that both substrates stabilize the β2β3 loop to a similar extent; differences in substrate selectivity arise primarily from differential binding affinities rather than differential loop closure.\",\n      \"method\": \"In vitro kinetic competition assays, varied ionic strength, amide H/D exchange monitored by mass spectrometry\",\n      \"journal\": \"Journal of inorganic biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic assays with kinetic measurements and structural HDX-MS in a single focused biochemical study\",\n      \"pmids\": [\"23787140\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Novel crystal structure of PHD2 enzyme complexed with a 2,8-diazaspiro[4.5]decan-1-one inhibitor reveals previously unobserved binding interactions: hydrogen bond with Arg322, π-cation interaction with Arg322, π-π stacking with Trp389 and His313, without the salt bridge to Arg383 seen in all prior complex structures.\",\n      \"method\": \"X-ray crystallography of PHD2-inhibitor complex, structure-activity relationship studies\",\n      \"journal\": \"Bioorganic & medicinal chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Weak — crystal structure is Tier 1, but single study and focused on inhibitor binding rather than mechanistic substrate work\",\n      \"pmids\": [\"24055079\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Endothelial PHD2 deficiency induces obliterative pulmonary vascular remodeling and PAH through suppression of caveolin-1 (Cav1), which augments nitrative stress via endothelial NOS activation. Genetic restoration of Cav1 in Egln1Tie2 mice normalized nitrative stress and reduced PAH, while NOS3 knockdown or superoxide dismutase mimetic also inhibited the vascular remodeling.\",\n      \"method\": \"Egln1Tie2 mouse model, Cav1 genetic rescue, endothelial NOS3 knockdown via CRISPR-Cas9 nanoparticle delivery, SOD mimetic treatment, hemodynamic measurements\",\n      \"journal\": \"The European respiratory journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple genetic and pharmacological interventions in vivo, single lab\",\n      \"pmids\": [\"35798360\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PHD2 deficiency in endothelial cells and carotid bodies increases ventilatory sensitivity to hypoxia and causes carotid body hyperplasia through HIF-2α but not HIF-1α. Inducible inactivation of HIF-2α impairs ventilatory acclimatization to chronic hypoxia, establishing the PHD2/HIF-2α enzyme-substrate couple as the key mediator of hypoxic ventilatory responses.\",\n      \"method\": \"Tamoxifen-inducible Phd2 knockout, HIF-1α and HIF-2α conditional knockout epistasis, plethysmography, BrdU proliferation assays in carotid bodies\",\n      \"journal\": \"The Journal of physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis in inducible models establishing PHD2/HIF-2α axis; replicated across constitutive and inducible deletion lines\",\n      \"pmids\": [\"26337139\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Inhibition of Egln1 systemically or in skeletal muscles protects mice against myocardial ischemia-reperfusion injury. The protection is mediated by a secreted factor: Egln1 loss causes accumulation of circulating α-ketoglutarate (αKG), which drives hepatic production and secretion of kynurenic acid (KYNA), which is necessary and sufficient to mediate cardiac ischemic protection.\",\n      \"method\": \"Somatic Egln1 gene deletion, pharmacological inhibition (PHD inhibitor), parabiosis experiments, metabolomics (αKG measurement), KYNA supplementation and depletion experiments\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — rigorous genetic and pharmacological models, parabiosis to define humoral factor, metabolomics to identify secreted αKG→KYNA axis; multiple orthogonal approaches\",\n      \"pmids\": [\"26919427\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Lactate, at physiological concentrations, directly binds to the catalytic domain of PHD2 in competition with α-ketoglutarate, inhibiting PHD2 activity and stabilizing HIF-1α. Lactate-induced IL-1β production was abolished in PHD2-deficient macrophages, identifying PHD2 as a direct metabolic sensor of lactate that connects chronic inflammation to energy metabolism.\",\n      \"method\": \"Direct binding assay (lactate to PHD2 catalytic domain), competitive inhibition kinetics vs. α-ketoglutarate, PHD2-deficient macrophage rescue experiments, in vivo adipocyte-specific LDHA deletion mouse model\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct binding/competitive inhibition assay with purified PHD2, genetic rescue in PHD2-deficient cells, in vivo mouse model; multiple orthogonal methods in single study\",\n      \"pmids\": [\"36064857\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SET7, a lysine monomethylase, catalyzes EGLN1 methylation at lysine 297, repressing EGLN1 prolyl hydroxylase activity toward HIF-1α. A methylation mimic mutant of EGLN1 loses the capability to suppress hypoxia signaling, leading to enhanced cell proliferation and increased oxygen consumption rate.\",\n      \"method\": \"In vitro methylation assay (SET7 acting on EGLN1), site-directed mutagenesis of K297, PHD2 activity assays, HIF-1α hydroxylation measurement, cell proliferation and metabolic assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic methylation assay plus site-directed mutagenesis with defined functional readout (prolyl hydroxylase activity); single study with multiple orthogonal methods\",\n      \"pmids\": [\"35452683\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"DAP5 (Death-associated protein 5) regulates HIF-1α abundance by enabling DAP5:eIF2β complex-dependent translation of PHD2 during hypoxia. DAP5 depletion caused a surge in HIF-1α levels due to reduced PHD2 translation, revealing a translational control mechanism that safeguards against excessive HIF-1α accumulation during hypoxia-associated protein synthesis repression.\",\n      \"method\": \"DAP5 siRNA knockdown, polysome profiling for PHD2 mRNA, co-IP of DAP5:eIF2β, HIF-1α protein measurement, PHD2 reporter constructs\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus translational assay and HIF-1α functional readout; single lab\",\n      \"pmids\": [\"29530922\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"EglN1/PHD2 accumulates on mitochondria under hypoxia via its β2β3 loop substrate-binding region, where it interacts with and prolyl-hydroxylates AMPKα under normoxia, causing their rapid dissociation and release from mitochondria. Under hypoxia, the EglN1-AMPKα interaction is maintained, and a CaMKK2-EglN1-AMPKα complex forms on mitochondria, activating AMPKα phosphorylation to maintain metabolic homeostasis and support breast tumor growth.\",\n      \"method\": \"Mitochondrial proteomic profiling, co-IP of EglN1-AMPKα on mitochondria, in vitro prolyl hydroxylation assay of AMPKα, β2β3 loop deletion mutants, CaMKK2 co-IP, xenograft tumor growth assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — novel mitochondrial substrate (AMPKα) identified by proteomics, in vitro hydroxylation assay, structural mutant validation of β2β3 loop, complex characterization; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"37661833\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Osteocyte PHD2 (Egln1) controls FGF23 production: conditional osteocyte-specific Phd2 deletion upregulates Fgf23 through HIF-1α-dependent mechanisms. PHD2-knockout osteocyte-like cells lose iron-mediated suppression of Fgf23 that cannot be compensated by PHD1 or PHD3, and in vivo CKD models show suppressed Phd1-3 consistent with FGF23 upregulation, linking the oxygen/iron-sensing PHD2 axis to phosphate homeostasis.\",\n      \"method\": \"Osteocyte-specific Phd2 conditional knockout, CRISPR Phd2-KO osteocyte cell lines, ATACseq/RNAseq, HIF1α blockade rescue, chronic kidney disease mouse model\",\n      \"journal\": \"Bone research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — osteocyte-specific KO with genomic and transcriptional analyses plus HIF1α rescue; single lab\",\n      \"pmids\": [\"36650133\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Induction of SM-20 (EGLN1) expression in PC12 cells leads to increased cytochrome c levels, accumulation of cytochrome c in the cytosol, and caspase-dependent cell death, establishing a mechanism by which SM-20 promotes neuronal apoptosis through the cytochrome c/caspase pathway.\",\n      \"method\": \"Doxycycline-inducible SM-20 PC12 cell line, subcellular fractionation of cytochrome c, caspase activity assays, caspase inhibitor (zVAD-FMK) rescue\",\n      \"journal\": \"Journal of neurochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — inducible expression system with subcellular fractionation and pharmacological rescue; single lab\",\n      \"pmids\": [\"12675908\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"PHD2 inhibition in cancer-associated fibroblasts (CAFs) phenocopies the effects of chronic hypoxia: PHD2 loss leads to HIF-1α stabilization, reduced αSMA and periostin expression, decreased myosin II activity, loss of contractile force, and reduced ECM remodeling. This deactivates CAFs and impairs CAF-mediated cancer cell invasion and metastasis. These effects require HIF-1α, as simultaneous HIF-1α depletion prevents the PHD2 loss phenotype.\",\n      \"method\": \"PHD2 RNAi and chemical inhibition (DMOG) in CAFs, orthotopic breast cancer model, αSMA/periostin expression analysis, traction force microscopy, HIF-1α siRNA epistasis\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic and pharmacological PHD2 loss-of-function with HIF-1α epistasis and in vivo tumor model; single lab\",\n      \"pmids\": [\"26323721\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PHD2 repression in macrophages is induced by angiopoietin-1 (ANG1)-mediated signaling during femoral artery occlusion. ANG1-dependent PHD2 repression initiates a feed-forward loop mediated by induction of the ANG receptor TIE2 in macrophages. TIE2 induction is required for proarteriogenic macrophage functions enabling collateral vessel formation.\",\n      \"method\": \"ANG1 blockade (soluble trap), Phd2 gene silencing in macrophages, macrophage depletion/transfer, TIE2 gene silencing, hindlimb ischemia model\",\n      \"journal\": \"EMBO molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic silencing and depletion strategies with defined ANG1-PHD2-TIE2 pathway; single lab\",\n      \"pmids\": [\"23616286\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Endothelial PHD2 deletion protects against renal ischemia-reperfusion injury by suppressing proinflammatory gene expression and inflammatory cell recruitment in a manner dependent on HIF-1 but not HIF-2. In vitro experiments implicated a humoral factor in the anti-inflammatory effects of endothelial PHD2/HIF-1 signaling.\",\n      \"method\": \"Endothelial-specific Phd2 deletion, double KO with HIF-1 or HIF-2, inducible endothelial Phd2 deletion in adult mice, renal ischemia-reperfusion injury model, inflammatory cell quantification\",\n      \"journal\": \"Journal of the American Society of Nephrology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with HIF isoform epistasis plus inducible deletion; single lab\",\n      \"pmids\": [\"31996410\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The gain-of-function Tibetan PHD2 D4E;C127S variant suppresses hypoxia-induced inflammatory responses in monocytes: PHD2D4E;C127S monocytes show reduced secretion of IL-6 and IL-1β, impaired chemotaxis, and downmodulation of RELA, JUN, STAT1, ATF2 and CXCR4. U937 monocytic cells engineered to express PHD2D4E;C127S confirmed these findings at the protein level under hypoxia. αKG supplementation (augmenting PHD2 activity) also diminished inflammatory responses in vitro and reduced leukocyte infiltration in lungs of hypoxia-exposed mice.\",\n      \"method\": \"Flow cytometry immunophenotyping of homozygous Tibetan PHD2 variant carriers, nCounter gene expression analysis, engineered U937 cell lines expressing Tibetan variant vs. WT PHD2, in vivo hypoxia mouse model with αKG supplementation\",\n      \"journal\": \"EBioMedicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ex vivo Tibetan cohort plus engineered cell line validation and in vivo rescue; single lab\",\n      \"pmids\": [\"34102396\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"EGLN1/PHD2 is an iron(II)- and 2-oxoglutarate-dependent dioxygenase that functions as the primary cellular oxygen sensor by prolyl-hydroxylating HIF-1α and HIF-2α (preferring the CODD site ~20-fold over NODD), targeting them for VHL-mediated ubiquitination and proteasomal degradation; beyond HIF-α, it also hydroxylates non-HIF substrates including filamin A (regulating dendritic spines), thyroid hormone receptor-α (regulating phospholamban and cardiac contractility), B55α/PP2A (regulating cell survival under nutrient stress), and AMPKα (on mitochondria during hypoxia to support metabolic homeostasis); its activity is modulated by multiple post-translational mechanisms including S125 phosphorylation by mTOR/P70S6K (activating) and PP2A/B55α-mediated dephosphorylation (inhibiting), K297 methylation by SET7 (inhibiting), persulfidation at Cys21/Cys33 by CBS-derived H2S (activating), oxidative disulfide-mediated homodimerization (inhibiting), and competitive inhibition by lactate at the α-KG binding site; PHD2 interacts with the HSP90 cochaperone p23 and FKBP38 via its N-terminal MYND zinc finger domain to link to the chaperone pathway, and cycles between cytoplasm and nucleus via CRM1-dependent export; in vivo, PHD2 is the dominant regulator of erythropoiesis, vascular normalization, arteriogenesis, ventilatory hypoxic response (through HIF-2α), immune regulation, and multiple organ-specific processes, and the Tibetan D4E/C127S haplotype represents a loss-of-function allele that impairs p23 binding and blunts HIF pathway activity to facilitate high-altitude adaptation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"EGLN1/PHD2 is an iron(II)- and 2-oxoglutarate-dependent prolyl hydroxylase that serves as the principal cellular oxygen sensor, hydroxylating specific prolyl residues on HIF-1\\u03b1 and HIF-2\\u03b1 to target them for proteolysis; loss-of-function studies establish PHD2 as the dominant HIF prolyl hydroxylase under normoxia and in vivo [#0, #4]. It preferentially hydroxylates the HIF-1\\u03b1 CODD site ~20-fold over NODD, a selectivity governed primarily by differential substrate binding affinity [#23]. Because PHD2 is itself transcribed from a hypoxia-responsive promoter directly bound by HIF-1, the enzyme operates within a negative-feedback loop that restrains the HIF response [#1], and additional layers of control include DAP5/eIF2\\u03b2-dependent translation of PHD2 mRNA during hypoxia [#30]. PHD2 activity is tuned by a dense array of post-translational and metabolic inputs: activating S125 phosphorylation by the mTOR/P70S6K axis opposed by PP2A-B55\\u03b1 dephosphorylation [#13], ERK phosphorylation that blocks HIF-1\\u03b1 binding [#20], inhibitory K297 methylation by SET7 [#29], activating persulfidation at Cys21/Cys33 by CBS-derived H2S [#21], inactivating oxidative disulfide homodimerization [#14], and direct competitive inhibition at the \\u03b1-ketoglutarate site by lactate [#28]. Beyond HIF-\\u03b1, PHD2 hydroxylates non-HIF substrates that connect it to diverse cellular programs: filamin A to control dendritic spine maturation [#12], thyroid hormone receptor-\\u03b1 to regulate phospholamban and cardiac stress tolerance [#15], the PP2A subunit B55\\u03b1 to govern apoptosis under glucose starvation [#19], and mitochondrial AMPK\\u03b1 within a CaMKK2-containing complex to maintain metabolic homeostasis in hypoxia [#31]. The enzyme links to the HSP90 chaperone pathway through its N-terminal MYND zinc finger, which binds a PXLE motif in the co-chaperones p23 and FKBP38 to facilitate HIF-\\u03b1 hydroxylation and regulate PHD2 stability [#7, #10], and it shuttles between cytoplasm and nucleus via CRM1-dependent export [#8]. In vivo, PHD2 is the master regulator of erythropoiesis and cardiac homeostasis [#4], tumor and ischemic vascular normalization and arteriogenesis [#5, #9], pulmonary vascular remodeling through HIF-2\\u03b1 [#16], and the hypoxic ventilatory response via the PHD2/HIF-2\\u03b1 couple in the carotid body [#26]. The Tibetan D4E/C127S haplotype is a loss-of-function allele that impairs p23 binding, blunting the HIF pathway and the hypoxic ventilatory/inflammatory response to facilitate high-altitude adaptation [#22, #37].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Before its enzymatic identity was known, EGLN1 (SM-20) was found to localize to mitochondria and drive neuronal apoptosis, establishing an early functional readout that would later be reconciled with its dioxygenase role.\",\n      \"evidence\": \"fusion-protein import assays, co-localization, and caspase-inhibitor rescue in NGF-deprived neurons and PC12 cells\",\n      \"pmids\": [\"11060309\", \"12675908\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"did not identify the enzymatic activity or molecular substrate\", \"relationship between pro-apoptotic activity and prolyl hydroxylase function unresolved\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Defining EGLN1 as the dominant cellular HIF prolyl hydroxylase answered which enzyme sets normoxic HIF-\\u03b1 levels and revealed site- and isoform-selectivity.\",\n      \"evidence\": \"isoform-specific siRNA knockdown with HIF-\\u03b1 hydroxylation site readouts across multiple cell types\",\n      \"pmids\": [\"15247232\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"kinetic basis of site selectivity not yet defined\", \"did not address regulation of PHD2 itself\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Identifying PHD2 as a direct HIF-1 target gene with a functional HRE explained how the oxygen-sensing system self-limits through negative feedback.\",\n      \"evidence\": \"promoter luciferase reporters, EMSA, and transcript analysis of the downstream CpG-island promoter\",\n      \"pmids\": [\"15563275\", \"16157596\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"mechanism of HIF-independent transcriptional repression by PHD2 not fully defined\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Conditional mouse knockout established PHD2 as the primary HIF prolyl hydroxylase in vivo and tied it to erythropoiesis and cardiac homeostasis.\",\n      \"evidence\": \"Cre-lox conditional Egln1 inactivation with phenotypic and gene-expression profiling\",\n      \"pmids\": [\"18096761\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"did not resolve which HIF isoform mediates each phenotype\", \"tissue-specific contributions not dissected\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Cell-type-specific PHD2 haplodeficiency revealed roles in endothelial vascular normalization and macrophage-driven arteriogenesis, including HIF-independent NF-\\u03baB-dependent outputs.\",\n      \"evidence\": \"Phd2(+/-) tumor and ischemia mouse models with HIF and NF-\\u03baB pathway epistasis and cytokine assays\",\n      \"pmids\": [\"19217150\", \"19477431\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"direct substrate underlying NF-\\u03baB/HIF-independent effects not identified\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Mapping the N-terminal MYND zinc finger as a protein-interaction module connected PHD2 to chaperone-pathway partners and to non-canonical trafficking control.\",\n      \"evidence\": \"peptide arrays, FRET, co-IP, fractionation for FKBP38 binding, plus CRM1-inhibition and importin-competition for nucleocytoplasmic shuttling\",\n      \"pmids\": [\"19546213\", \"19631610\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"functional consequence of nuclear pool of PHD2 unresolved\", \"FKBP38-dependent stability mechanism not linked to specific physiology\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Biochemical and structural studies quantified CODD-over-NODD selectivity and defined inhibitor-binding chemistry of the catalytic site.\",\n      \"evidence\": \"kinetic competition assays, HDX-MS, and X-ray crystallography of PHD2-inhibitor complexes\",\n      \"pmids\": [\"23787140\", \"24055079\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"structural basis for non-HIF substrate recognition not addressed\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defining the PXLE motif shared by p23 and FKBP38, and later by HSP90, mechanistically linked PHD2 to the HSP90 chaperone machinery as a determinant of HIF-\\u03b1 hydroxylation.\",\n      \"evidence\": \"co-IP, PXLE motif identification, and p23 knockdown with HIF-1\\u03b1 readouts; ANG1-TIE2 feed-forward loop in macrophages\",\n      \"pmids\": [\"23413029\", \"23616286\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"how chaperone binding mechanistically enhances catalysis not defined\", \"single-lab characterization of the p23 dependency\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identifying filamin A and cancer-associated fibroblast deactivation extended PHD2 function to HIF-independent substrate hydroxylation and tumor-stroma mechanics.\",\n      \"evidence\": \"site-directed mutagenesis of FLNA prolines, ubiquitination and spine imaging; CAF traction-force microscopy with HIF-1\\u03b1 epistasis\",\n      \"pmids\": [\"26972007\", \"26323721\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"physiological contexts of FLNA hydroxylation beyond neurons not mapped\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"A wave of in vivo and biochemical studies expanded the PHD2 substrate/regulatory repertoire (TR-\\u03b1, oxidative dimerization, S125 phosphorylation) and dissected organ-specific HIF isoform dependencies in pulmonary, ventilatory, ischemic, and metabolic settings.\",\n      \"evidence\": \"double conditional KOs (Phd2/Phd3, Egln1/Hif1a, Egln1/Hif2a), in vitro hydroxylation of TR-\\u03b1, cysteine mutagenesis, phospho-specific assays, parabiosis and metabolomics, Seahorse metabolic profiling\",\n      \"pmids\": [\"26075818\", \"26740011\", \"28199842\", \"27143681\", \"26337139\", \"26919427\", \"27795296\", \"27919930\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"integration of competing post-translational inputs in a single cell type unresolved\", \"relative contribution of each non-HIF substrate to organ phenotypes unclear\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Discovery of DAP5/eIF2\\u03b2-dependent translation of PHD2 mRNA added a translational safeguard limiting HIF-1\\u03b1 accumulation during hypoxic protein-synthesis repression.\",\n      \"evidence\": \"DAP5 knockdown, polysome profiling of PHD2 mRNA, and DAP5:eIF2\\u03b2 co-IP\",\n      \"pmids\": [\"29530922\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"single-lab characterization\", \"in vivo relevance of translational control not established\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Persulfidation by CBS-derived H2S and genetic confirmation of the Tibetan allele as a p23-pathway loss-of-function variant clarified how PHD2 activity is metabolically and evolutionarily tuned for hypoxic adaptation.\",\n      \"evidence\": \"persulfidation assays with Cys21/Cys33 mutagenesis and zebrafish rescue; Tibetan Phd2 and p23-mutant knockin mice with plethysmography; endothelial Cav1/NOS rescue in renal/pulmonary injury models\",\n      \"pmids\": [\"32937467\", \"32414920\", \"35798360\", \"31996410\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"interplay between persulfidation and other cysteine-based redox regulation not resolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Direct lactate competition at the \\u03b1KG site and SET7-mediated K297 methylation revealed PHD2 as a direct metabolic and epigenetic sensor coupling energy state to HIF signaling and inflammation.\",\n      \"evidence\": \"direct binding/competition kinetics with purified PHD2, in vitro methylation assays with K297 mutants, and PHD2-deficient macrophage rescue; Tibetan-variant monocyte immunophenotyping\",\n      \"pmids\": [\"36064857\", \"35452683\", \"34102396\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"quantitative hierarchy among lactate, methylation, and phosphorylation inputs unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identification of mitochondrial AMPK\\u03b1 as a substrate within a CaMKK2-EglN1-AMPK\\u03b1 complex and osteocyte control of FGF23 placed PHD2 at the interface of organelle-localized metabolic signaling and systemic phosphate/iron homeostasis.\",\n      \"evidence\": \"mitochondrial proteomics, in vitro AMPK\\u03b1 hydroxylation, \\u03b22\\u03b23 loop deletion mutants and xenografts; osteocyte-specific Phd2 KO with ATAC/RNAseq and HIF-1\\u03b1 rescue\",\n      \"pmids\": [\"37661833\", \"36650133\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"structural basis of mitochondrial recruitment via \\u03b22\\u03b23 loop not solved\", \"generality of mitochondrial AMPK\\u03b1 hydroxylation across tissues untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the many competing post-translational, redox, and metabolic regulators of PHD2 are integrated in real time within a single cell, and how substrate choice between HIF-\\u03b1 and the growing set of non-HIF substrates is determined, remain open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"no unified model reconciling phosphorylation, methylation, persulfidation, dimerization, and lactate inhibition\", \"substrate-selection rules for non-HIF targets undefined\", \"structural determinants of organelle-specific PHD2 activity unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016491\", \"supporting_discovery_ids\": [0, 23, 12, 15, 19, 31]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 12, 15, 19, 31]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 23]},\n      {\"term_id\": \"GO:0140299\", \"supporting_discovery_ids\": [28, 21]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [3, 7, 31]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [7, 8]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [0, 4, 26]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [17, 27, 28, 31]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [9, 28, 36, 37]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [3, 33, 19]}\n    ],\n    \"complexes\": [\"CaMKK2-EglN1-AMPK\\u03b1 mitochondrial complex\"],\n    \"partners\": [\"HIF1A\", \"EPAS1\", \"FKBP38\", \"PTGES3\", \"HSP90\", \"FLNA\", \"THRA\", \"PPP2R2A\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}