{"gene":"PYCR2","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":2016,"finding":"PYCR1 and PYCR2 were identified as components of RRM2B complexes by mass spectrometry after large-scale purification of Flag-tagged RRM2B. Silencing of both PYCR1 and PYCR2 completely abolished the anti-oxidation activity of RRM2B, demonstrating functional collaboration between these metabolic enzymes in response to oxidative stress.","method":"Flag-tag affinity purification + mass spectrometry (complex identification); shRNA knockdown with oxidative stress assay (functional epistasis)","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP/MS for complex identification plus functional epistasis via knockdown, single lab, two orthogonal methods","pmids":["26733354"],"is_preprint":false},{"year":2015,"finding":"Disease-associated PYCR2 missense variants (p.Arg119Cys and p.Arg251Cys) retain mitochondrial localization but are less stable than wild-type protein. PYCR2 loss of function (CRISPR-Cas9 knockout) leads to decreased mitochondrial membrane potential and increased susceptibility to apoptosis under oxidative stress.","method":"CRISPR-Cas9 knockout cell line; transfection of mutant cDNAs with localization imaging; mitochondrial membrane potential assay; apoptosis assay under oxidative stress","journal":"American journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO with defined cellular phenotypes, mutant localization experiments, single lab, multiple orthogonal methods","pmids":["25865492"],"is_preprint":false},{"year":2016,"finding":"PYCR2 missense mutations identified in patients impair protein multimerization, establishing that proper oligomeric assembly is required for normal PYCR2 function.","method":"Biochemical analysis of patient-derived mutations; protein multimerization assay","journal":"Annals of neurology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, limited methodological detail in abstract regarding multimerization assay","pmids":["27130255"],"is_preprint":false},{"year":2020,"finding":"Crystal structure of the PYCR2 apo-enzyme was determined. A disease-associated p.Gly249Val mutation lies at the dimer interface and lowers enzymatic activity. Loss of PYCR2 upregulates SHMT2, which increases cerebral glycine levels; SHMT2 knockdown partially rescued axonal beading and neurite length defects in Pycr2 knockout neurons. Loss of PYCR2 also depletes PYCR1 levels in neural lineages.","method":"Crystal structure determination; Pycr2 knockout mouse model phenotyping; in situ neurotransmitter quantification in brain; SHMT2 knockdown rescue experiments in cultured neurons; enzymatic activity assays","journal":"Neuron","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure, in vitro enzymatic assay, KO mouse model, genetic epistasis via SHMT2 knockdown rescue, multiple orthogonal methods in one rigorous study","pmids":["32330411"],"is_preprint":false},{"year":2021,"finding":"PYCR2 wild-type and disease variants (R119C and R251C) were kinetically characterized; wild-type enzyme follows a sequential binding mechanism with L-P5C binding before NAD(P)H and NAD(P)+ releasing before L-Pro. Both R119C and R251C variants are catalytically impaired: R119C has 40- or 366-fold lower catalytic efficiency (with NADPH or NADH respectively), while R251C has 7- or 26-fold lower catalytic efficiency. R251C also exhibits a pronounced folding defect by thermostability and circular dichroism measurements.","method":"Steady-state kinetic measurements; thermostability assay; circular dichroism spectroscopy; in vitro enzyme assay with purified recombinant proteins","journal":"Archives of biochemistry and biophysics","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified protein, kinetic mechanism established by substrate-order experiments, mutagenesis of disease variants, multiple orthogonal biophysical methods","pmids":["33771508"],"is_preprint":false},{"year":2021,"finding":"Both PYCR1 and PYCR2 localize to mitochondria in fibroblasts. Both proteins complement loss of yeast Pro3 (the P5C-to-proline reductase), confirming their activity as P5C reductases. Pycr1;Pycr2 double-mutant mice are sub-viable and worse than either single mutant, indicating largely redundant functions in proline biosynthesis.","method":"Mitochondrial localization by cell fractionation/imaging; yeast complementation assay; mouse double-mutant genetic epistasis; serum/tissue proline measurements","journal":"Genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast complementation (functional reconstitution), direct localization experiments, genetic epistasis in double-mutant mice, single lab","pmids":["33734376"],"is_preprint":false},{"year":2022,"finding":"E4B ubiquitin E3 ligase ubiquitinates PYCR2 both in vitro and in HEK293 cells, forming K48-linked polyubiquitin chains on PYCR2 to promote its proteasomal degradation. E4B interacts with PYCR2 via its variable region.","method":"In vitro ubiquitination assay; co-immunoprecipitation in HEK293 cells; K48 linkage-specific ubiquitin chain analysis; domain mapping of E4B variable region","journal":"Frontiers in cell and developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro ubiquitination assay plus cell-based co-IP with linkage typing, single lab, two orthogonal methods","pmids":["35669517"],"is_preprint":false},{"year":2022,"finding":"HLD10-associated PYCR2 mutations R119C and R251C cause formation of abnormally large mitochondria in oligodendroglial cells (FBD-102b), with increased mitochondrial fusion and decreased fission, and decreased mitochondrial activity. Cells expressing these mutants fail to undergo morphological differentiation upon induction, unlike wild-type PYCR2-expressing cells.","method":"Transfection of mutant and wild-type PYCR2 into oligodendroglial cell line; mitochondrial morphology imaging; mitochondrial fusion/fission activity assays; differentiation marker expression by western blot","journal":"Neurology international","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — defined cellular phenotype with loss-of-function mutants in relevant cell type, multiple orthogonal methods, single lab","pmids":["36548190"],"is_preprint":false},{"year":2021,"finding":"ASFV E199L protein interacts with PYCR2 (identified by co-immunoprecipitation coupled with mass spectrometry) and downregulates PYCR2 expression, resulting in autophagy activation.","method":"Co-immunoprecipitation coupled with mass spectrometry; western blot for PYCR2 expression; autophagy assays in Vero and HEK-293T cells","journal":"Virologica Sinica","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP/MS in a viral context, single lab, limited mechanistic follow-up on PYCR2 itself","pmids":["33830435"],"is_preprint":false},{"year":2023,"finding":"c-Myc binds to the PYCR2 promoter and transcriptionally upregulates PYCR2 expression, as demonstrated by luciferase reporter assay and western blot in breast cancer cells.","method":"Luciferase reporter assay with PYCR2 promoter; western blot; chromatin binding analysis","journal":"The international journal of biochemistry & cell biology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — luciferase reporter assay for transcriptional regulation, single lab, single method for the transcriptional mechanism","pmids":["38101533"],"is_preprint":false},{"year":2023,"finding":"ALKBH5 promotes PYCR2 expression (and PYCR2-mediated proline synthesis), while PYCR2 in turn promotes ALKBH5 expression through the AMPK/mTOR pathway, forming a positive feedback loop in GBM cells. Proline supplementation rescued AMPK/mTOR activation and proneural-mesenchymal transition after PYCR2 silencing.","method":"siRNA/shRNA knockdown; western blot; proline supplementation rescue experiment; AMPK/mTOR pathway analysis","journal":"Journal of Cancer","confidence":"Low","confidence_rationale":"Tier 3 / Weak — pathway epistasis by knockdown and rescue, single lab, limited mechanistic resolution from abstract alone","pmids":["37325047"],"is_preprint":false},{"year":2025,"finding":"PFDN2 physically interacts with PYCR2 (co-immunoprecipitation; cytoplasmic colocalization by immunofluorescence) and stabilizes PYCR2 protein by limiting proteasome-dependent degradation, as shown by cycloheximide chase and MG132 rescue experiments. PYCR2 activity is required downstream of PFDN2 to activate Wnt/β-catenin signaling in colorectal cancer cells.","method":"Co-immunoprecipitation; immunofluorescence colocalization; cycloheximide chase assay; MG132 proteasome inhibitor rescue; TOP/FOPflash reporter assay; rescue experiments with PYCR2 re-expression","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP, protein stability assays with inhibitor rescue, pathway reporter assay, multiple orthogonal methods, single lab","pmids":["41656306"],"is_preprint":false}],"current_model":"PYCR2 is a mitochondria-localized enzyme that catalyzes the final step of proline biosynthesis (reduction of L-Δ1-pyrroline-5-carboxylate to L-proline via a sequential NAD(P)H-dependent mechanism); disease-associated mutations impair catalysis and/or protein folding/multimerization, and loss of PYCR2 causes neurodegeneration by upregulating SHMT2, elevating cerebral glycine, disrupting mitochondrial dynamics in oligodendrocytes, and depleting PYCR1 in neural lineages; PYCR2 protein stability is regulated by E4B-mediated K48-linked ubiquitination and by PFDN2-dependent protection from proteasomal degradation; PYCR2 also functionally collaborates with RRM2B to protect cells from oxidative stress."},"narrative":{"mechanistic_narrative":"PYCR2 is a mitochondrial NAD(P)H-dependent reductase that catalyzes the terminal step of proline biosynthesis, converting L-Δ1-pyrroline-5-carboxylate (L-P5C) to L-proline through an ordered sequential mechanism in which L-P5C binds before NAD(P)H and NAD(P)+ is released before L-proline [PMID:32330411, PMID:33771508, PMID:33734376]. The enzyme is active as an oligomer—a crystal structure of the apo-enzyme places the disease-associated p.Gly249Val substitution at the dimer interface where it lowers catalytic activity, and patient missense mutations impair multimeric assembly [PMID:27130255, PMID:32330411]. PYCR2 functions largely redundantly with PYCR1 as a P5C reductase, both localizing to mitochondria and both complementing loss of the yeast P5C-to-proline reductase, with double-mutant mice more severely affected than either single mutant [PMID:33734376]. Loss of PYCR2 causes neurodegeneration: it upregulates SHMT2 and elevates cerebral glycine, depletes PYCR1 in neural lineages, and impairs neuronal morphology, while SHMT2 knockdown partially rescues the axonal and neurite defects [PMID:32330411]. Biallelic PYCR2 variants such as p.Arg119Cys and p.Arg251Cys—which retain mitochondrial targeting but are catalytically impaired and, for R251C, also misfolded—underlie hypomyelinating leukodystrophy, with mutant proteins driving abnormal mitochondrial fusion/fission balance and blocking oligodendroglial differentiation [PMID:25865492, PMID:33771508, PMID:36548190]. PYCR2 abundance is controlled post-translationally by E4B-mediated K48-linked polyubiquitination targeting it for proteasomal degradation and by PFDN2, which binds PYCR2 and protects it from degradation [PMID:35669517, PMID:41656306]. Functionally, PYCR2 collaborates with RRM2B to confer resistance to oxidative stress, and its loss lowers mitochondrial membrane potential and sensitizes cells to apoptosis [PMID:26733354, PMID:25865492].","teleology":[{"year":2015,"claim":"Establishing that PYCR2 missense variants disrupt protein stability and cellular fitness rather than mislocalization linked the gene to disease through a loss-of-function mechanism.","evidence":"CRISPR-Cas9 knockout cell line with mitochondrial membrane potential and apoptosis assays, plus mutant cDNA localization imaging","pmids":["25865492"],"confidence":"Medium","gaps":["Did not resolve whether instability reflects catalytic or folding defects","No structural basis for variant destabilization"]},{"year":2016,"claim":"Identifying impaired multimerization in patient mutants connected oligomeric assembly to PYCR2 function, framing assembly as a disease-relevant property.","evidence":"Biochemical multimerization assay of patient-derived mutations","pmids":["27130255"],"confidence":"Low","gaps":["Limited methodological detail on the multimerization assay","Oligomeric state not linked to catalytic output here","No structural model"]},{"year":2016,"claim":"Placing PYCR1/PYCR2 within RRM2B complexes and showing their joint requirement for RRM2B anti-oxidation activity revealed a role beyond proline synthesis in oxidative stress defense.","evidence":"Flag-RRM2B affinity purification with mass spectrometry plus shRNA double-knockdown under oxidative stress","pmids":["26733354"],"confidence":"Medium","gaps":["Direct physical contact between PYCR2 and RRM2B not demonstrated","Mechanism of anti-oxidation contribution unresolved"]},{"year":2020,"claim":"A crystal structure plus a knockout mouse defined the catalytic architecture and the in vivo neurodegenerative cascade, showing loss of PYCR2 elevates glycine via SHMT2 and depletes PYCR1.","evidence":"Apo-enzyme crystal structure, Pycr2 knockout mouse phenotyping, brain neurotransmitter quantification, and SHMT2-knockdown rescue in cultured neurons","pmids":["32330411"],"confidence":"High","gaps":["Mechanism linking SHMT2 upregulation to PYCR2 loss not defined","How PYCR1 depletion is triggered unclear","No holo/substrate-bound structure"]},{"year":2021,"claim":"Steady-state kinetics established the ordered catalytic mechanism and quantified how disease variants impair catalysis versus folding, distinguishing two failure modes.","evidence":"Steady-state kinetic measurements, thermostability and circular dichroism on purified recombinant wild-type and R119C/R251C proteins","pmids":["33771508"],"confidence":"High","gaps":["Substrate-bound structural states not captured","Physiological cofactor preference (NADPH vs NADH) in vivo unresolved"]},{"year":2021,"claim":"Yeast complementation and double-mutant mice confirmed PYCR2 as a bona fide P5C reductase that is largely functionally redundant with PYCR1.","evidence":"Yeast Pro3 complementation, mitochondrial localization, Pycr1;Pycr2 double-mutant mouse genetics and tissue proline measurements","pmids":["33734376"],"confidence":"Medium","gaps":["Non-redundant, tissue-specific roles of the two enzymes not delineated","Quantitative contribution of each isozyme to proline pools unresolved"]},{"year":2021,"claim":"A viral interactor was found to downregulate PYCR2 and trigger autophagy, hinting at PYCR2 levels influencing autophagic signaling.","evidence":"Co-IP/MS of ASFV E199L with PYCR2, western blot and autophagy assays in Vero and HEK-293T cells","pmids":["33830435"],"confidence":"Low","gaps":["Single Co-IP/MS without reciprocal validation","Direct causal link between PYCR2 loss and autophagy not isolated"]},{"year":2022,"claim":"Identifying E4B-mediated K48 polyubiquitination defined a proteasomal route controlling PYCR2 abundance.","evidence":"In vitro ubiquitination assay, co-IP in HEK293 cells, K48 linkage typing, and E4B variable-region domain mapping","pmids":["35669517"],"confidence":"Medium","gaps":["Physiological contexts regulating E4B–PYCR2 not defined","Deubiquitinase counterpart unknown"]},{"year":2022,"claim":"Showing that R119C/R251C drive abnormal mitochondrial fusion/fission and block oligodendroglial differentiation connected variant biochemistry to the cellular pathology of hypomyelinating leukodystrophy.","evidence":"Transfection of wild-type/mutant PYCR2 into FBD-102b oligodendroglial cells with mitochondrial morphology, fusion/fission and differentiation-marker readouts","pmids":["36548190"],"confidence":"Medium","gaps":["Molecular link between reductase deficiency and fission/fusion machinery unclear","Overexpression system may not reflect endogenous stoichiometry"]},{"year":2023,"claim":"Transcriptional and m6A-associated regulators (c-Myc, ALKBH5) were placed upstream of PYCR2 in cancer, embedding it in proline-driven oncogenic circuits.","evidence":"Luciferase promoter reporter and western blot (c-Myc); knockdown, proline rescue and AMPK/mTOR analysis (ALKBH5 feedback loop) in breast and glioblastoma cells","pmids":["38101533","37325047"],"confidence":"Low","gaps":["Single-method transcriptional evidence per study","Direct vs indirect regulation not fully separated","Generality across tumor types untested"]},{"year":2025,"claim":"PFDN2 was shown to bind and stabilize PYCR2 against proteasomal turnover, with PYCR2 activity required for Wnt/β-catenin signaling, defining a stabilizing partner opposing E4B-driven degradation.","evidence":"Reciprocal co-IP, immunofluorescence colocalization, cycloheximide chase, MG132 rescue, and TOP/FOPflash reporter with PYCR2 re-expression in colorectal cancer cells","pmids":["41656306"],"confidence":"Medium","gaps":["How PFDN2 protects PYCR2 mechanistically (shielding vs competition with E4B) unknown","Link between proline metabolism and Wnt activation not mechanistically resolved"]},{"year":null,"claim":"How PYCR2's enzymatic proline output is mechanistically coupled to its downstream non-metabolic phenotypes—mitochondrial dynamics, oligodendrocyte differentiation, oxidative-stress resistance and Wnt/AMPK-mTOR signaling—remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No substrate-bound structure to explain variant-specific defects","Causal chain from proline depletion to mitochondrial morphology changes undefined","In vivo relevance of cancer-associated regulatory loops untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016491","term_label":"oxidoreductase activity","supporting_discovery_ids":[3,4,5]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[4,5]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[1,5]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[11]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[3,4,5]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[6,11]}],"complexes":[],"partners":["RRM2B","PYCR1","E4B","PFDN2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q96C36","full_name":"Pyrroline-5-carboxylate reductase 2","aliases":[],"length_aa":320,"mass_kda":33.6,"function":"Oxidoreductase that catalyzes the last step in proline biosynthesis, which corresponds to the reduction of pyrroline-5-carboxylate to L-proline using NAD(P)H (PubMed:23024808, PubMed:2722838, PubMed:6894153). At physiologic concentrations, has higher specific activity in the presence of NADH (PubMed:23024808, PubMed:2722838, PubMed:6894153). Involved in cellular response to oxidative stress (PubMed:25865492). In some cell types, such as erythrocytes, its primary function may be the generation of NADP(+) (PubMed:2722838, PubMed:6894153)","subcellular_location":"Cytoplasm; Mitochondrion","url":"https://www.uniprot.org/uniprotkb/Q96C36/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PYCR2","classification":"Not Classified","n_dependent_lines":6,"n_total_lines":1208,"dependency_fraction":0.004966887417218543},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CDC16","stoichiometry":10.0},{"gene":"CDC27","stoichiometry":4.0},{"gene":"NUCKS1","stoichiometry":4.0},{"gene":"ANAPC16","stoichiometry":0.2},{"gene":"ANAPC2","stoichiometry":0.2},{"gene":"ANAPC4","stoichiometry":0.2},{"gene":"CDC23","stoichiometry":0.2},{"gene":"CLTA","stoichiometry":0.2},{"gene":"HIST2H2BE","stoichiometry":0.2},{"gene":"HMGA1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/PYCR2","total_profiled":1310},"omim":[{"mim_id":"616420","title":"LEUKODYSTROPHY, HYPOMYELINATING, 10; HLD10","url":"https://www.omim.org/entry/616420"},{"mim_id":"616408","title":"PYRROLINE-5-CARBOXYLATE REDUCTASE-LIKE; PYCRL","url":"https://www.omim.org/entry/616408"},{"mim_id":"616406","title":"PYRROLINE-5-CARBOXYLATE REDUCTASE 2; PYCR2","url":"https://www.omim.org/entry/616406"},{"mim_id":"312080","title":"PELIZAEUS-MERZBACHER DISEASE; PMD","url":"https://www.omim.org/entry/312080"},{"mim_id":"179035","title":"PYRROLINE-5-CARBOXYLATE REDUCTASE 1; PYCR1","url":"https://www.omim.org/entry/179035"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Mitochondria","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PYCR2"},"hgnc":{"alias_symbol":["P5CR2"],"prev_symbol":[]},"alphafold":{"accession":"Q96C36","domains":[{"cath_id":"3.40.50.720","chopping":"2-164","consensus_level":"high","plddt":96.9529,"start":2,"end":164},{"cath_id":"1.10.3730.10","chopping":"175-256","consensus_level":"medium","plddt":98.2705,"start":175,"end":256}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96C36","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96C36-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96C36-F1-predicted_aligned_error_v6.png","plddt_mean":89.56},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PYCR2","jax_strain_url":"https://www.jax.org/strain/search?query=PYCR2"},"sequence":{"accession":"Q96C36","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96C36.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96C36/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96C36"}},"corpus_meta":[{"pmid":"26733354","id":"PMC_26733354","title":"PYCR1 and PYCR2 Interact and Collaborate with RRM2B to Protect Cells from Overt Oxidative Stress.","date":"2016","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/26733354","citation_count":66,"is_preprint":false},{"pmid":"25865492","id":"PMC_25865492","title":"Mutations in PYCR2, Encoding Pyrroline-5-Carboxylate Reductase 2, Cause Microcephaly and Hypomyelination.","date":"2015","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/25865492","citation_count":64,"is_preprint":false},{"pmid":"33830435","id":"PMC_33830435","title":"African Swine Fever Virus Protein E199L Promotes Cell Autophagy through the Interaction of PYCR2.","date":"2021","source":"Virologica Sinica","url":"https://pubmed.ncbi.nlm.nih.gov/33830435","citation_count":38,"is_preprint":false},{"pmid":"27130255","id":"PMC_27130255","title":"PYCR2 Mutations cause a lethal syndrome of microcephaly and failure to thrive.","date":"2016","source":"Annals of neurology","url":"https://pubmed.ncbi.nlm.nih.gov/27130255","citation_count":36,"is_preprint":false},{"pmid":"32330411","id":"PMC_32330411","title":"Loss of PYCR2 Causes Neurodegeneration by Increasing Cerebral Glycine Levels via SHMT2.","date":"2020","source":"Neuron","url":"https://pubmed.ncbi.nlm.nih.gov/32330411","citation_count":30,"is_preprint":false},{"pmid":"27860360","id":"PMC_27860360","title":"Homozygous variants in pyrroline-5-carboxylate reductase 2 (PYCR2) in patients with progressive microcephaly and hypomyelinating leukodystrophy.","date":"2016","source":"American journal of medical genetics. Part A","url":"https://pubmed.ncbi.nlm.nih.gov/27860360","citation_count":21,"is_preprint":false},{"pmid":"36308281","id":"PMC_36308281","title":"Targeting PYCR2 inhibits intraperitoneal metastatic tumors of mouse colorectal cancer in a proline-independent approach.","date":"2022","source":"Cancer science","url":"https://pubmed.ncbi.nlm.nih.gov/36308281","citation_count":14,"is_preprint":false},{"pmid":"33771508","id":"PMC_33771508","title":"Disease variants of human Δ1-pyrroline-5-carboxylate reductase 2 (PYCR2).","date":"2021","source":"Archives of biochemistry and biophysics","url":"https://pubmed.ncbi.nlm.nih.gov/33771508","citation_count":13,"is_preprint":false},{"pmid":"33734376","id":"PMC_33734376","title":"Genetic analysis of Pycr1 and Pycr2 in mice.","date":"2021","source":"Genetics","url":"https://pubmed.ncbi.nlm.nih.gov/33734376","citation_count":11,"is_preprint":false},{"pmid":"37708598","id":"PMC_37708598","title":"PYCR2 promotes growth and aerobic glycolysis in human liver cancer by regulating the AKT signaling pathway.","date":"2023","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/37708598","citation_count":9,"is_preprint":false},{"pmid":"37325047","id":"PMC_37325047","title":"ALKBH5-PYCR2 Positive Feedback Loop Promotes Proneural-Mesenchymal Transition Via Proline Synthesis In GBM.","date":"2023","source":"Journal of Cancer","url":"https://pubmed.ncbi.nlm.nih.gov/37325047","citation_count":8,"is_preprint":false},{"pmid":"38101533","id":"PMC_38101533","title":"PYCR2, induced by c-Myc, promotes the invasiveness and metastasis of breast cancer by activating AKT signalling pathway.","date":"2023","source":"The international journal of biochemistry & cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/38101533","citation_count":7,"is_preprint":false},{"pmid":"35669517","id":"PMC_35669517","title":"Differential Degradation of TRA2A and PYCR2 Mediated by Ubiquitin E3 Ligase E4B.","date":"2022","source":"Frontiers in cell and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/35669517","citation_count":5,"is_preprint":false},{"pmid":"34055512","id":"PMC_34055512","title":"PYCR2 Mutation Causing Hypomyelination and Microcephaly in an Indian Child.","date":"2021","source":"Cureus","url":"https://pubmed.ncbi.nlm.nih.gov/34055512","citation_count":5,"is_preprint":false},{"pmid":"34037307","id":"PMC_34037307","title":"Expanding the genotypic spectrum of PYCR2 and a common ancestry in Thai patients with hypomyelinating leukodystrophy 10.","date":"2021","source":"American journal of medical genetics. Part A","url":"https://pubmed.ncbi.nlm.nih.gov/34037307","citation_count":5,"is_preprint":false},{"pmid":"36548190","id":"PMC_36548190","title":"Hypomyelinating Leukodystrophy 10 (HLD10)-Associated Mutations of PYCR2 Form Large Size Mitochondria, Inhibiting Oligodendroglial Cell Morphological Differentiation.","date":"2022","source":"Neurology international","url":"https://pubmed.ncbi.nlm.nih.gov/36548190","citation_count":3,"is_preprint":false},{"pmid":"37329534","id":"PMC_37329534","title":"LncRNA MALAT1 regulates growth of carcinoma of the lung through modulating miR-338-3p/PYCR2 axis.","date":"2023","source":"Cellular and molecular biology (Noisy-le-Grand, France)","url":"https://pubmed.ncbi.nlm.nih.gov/37329534","citation_count":2,"is_preprint":false},{"pmid":"38709052","id":"PMC_38709052","title":"Exploring metabolic alterations in PYCR2 deficiency: Unveiling pathways and clinical presentations of hypomyelinating leukodystrophy 10.","date":"2024","source":"American journal of medical genetics. Part A","url":"https://pubmed.ncbi.nlm.nih.gov/38709052","citation_count":1,"is_preprint":false},{"pmid":"37228935","id":"PMC_37228935","title":"Mutation in PYCR2 gene and hypomyelinating leukodystrophy in children: a case report study.","date":"2023","source":"Annals of medicine and surgery (2012)","url":"https://pubmed.ncbi.nlm.nih.gov/37228935","citation_count":1,"is_preprint":false},{"pmid":"41331125","id":"PMC_41331125","title":"LINC02878/ZNF282/PYCR2 axis promotes proline synthesis and tumor progression in colorectal cancer.","date":"2025","source":"Cellular and molecular life sciences : CMLS","url":"https://pubmed.ncbi.nlm.nih.gov/41331125","citation_count":1,"is_preprint":false},{"pmid":"39808866","id":"PMC_39808866","title":"Activation of mTOR/HK2 signaling mitigates effects of PYCR2 depletion in colorectal cells.","date":"2025","source":"Tissue & cell","url":"https://pubmed.ncbi.nlm.nih.gov/39808866","citation_count":1,"is_preprint":false},{"pmid":"37141741","id":"PMC_37141741","title":"Pyrroline-5-carboxylate reductase 2 (PYCR2) deficiency causes hereditary spastic paraplaegia in late childhood.","date":"2023","source":"European journal of paediatric neurology : EJPN : official journal of the European Paediatric Neurology Society","url":"https://pubmed.ncbi.nlm.nih.gov/37141741","citation_count":0,"is_preprint":false},{"pmid":"42155340","id":"PMC_42155340","title":"PYCR2 contributes to sunitinib resistance in hepatocellular carcinoma by activating the PPAR signaling pathway.","date":"2026","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/42155340","citation_count":0,"is_preprint":false},{"pmid":"41656306","id":"PMC_41656306","title":"PFDN2 stabilizes PYCR2 to activate Wnt/β-catenin signaling and promote colorectal cancer progression.","date":"2026","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/41656306","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.08.29.610420","title":"Comprehensive profiling of migratory primordial germ cells reveals niche-specific differences in non-canonical Wnt and Nodal-Lefty signaling in anterior vs posterior migrants","date":"2024-08-30","source":"bioRxiv","url":"https://doi.org/10.1101/2024.08.29.610420","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":15441,"output_tokens":3141,"usd":0.046719,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10559,"output_tokens":3849,"usd":0.07451,"stage2_stop_reason":"end_turn"},"total_usd":0.121229,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2016,\n      \"finding\": \"PYCR1 and PYCR2 were identified as components of RRM2B complexes by mass spectrometry after large-scale purification of Flag-tagged RRM2B. Silencing of both PYCR1 and PYCR2 completely abolished the anti-oxidation activity of RRM2B, demonstrating functional collaboration between these metabolic enzymes in response to oxidative stress.\",\n      \"method\": \"Flag-tag affinity purification + mass spectrometry (complex identification); shRNA knockdown with oxidative stress assay (functional epistasis)\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP/MS for complex identification plus functional epistasis via knockdown, single lab, two orthogonal methods\",\n      \"pmids\": [\"26733354\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Disease-associated PYCR2 missense variants (p.Arg119Cys and p.Arg251Cys) retain mitochondrial localization but are less stable than wild-type protein. PYCR2 loss of function (CRISPR-Cas9 knockout) leads to decreased mitochondrial membrane potential and increased susceptibility to apoptosis under oxidative stress.\",\n      \"method\": \"CRISPR-Cas9 knockout cell line; transfection of mutant cDNAs with localization imaging; mitochondrial membrane potential assay; apoptosis assay under oxidative stress\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with defined cellular phenotypes, mutant localization experiments, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"25865492\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PYCR2 missense mutations identified in patients impair protein multimerization, establishing that proper oligomeric assembly is required for normal PYCR2 function.\",\n      \"method\": \"Biochemical analysis of patient-derived mutations; protein multimerization assay\",\n      \"journal\": \"Annals of neurology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, limited methodological detail in abstract regarding multimerization assay\",\n      \"pmids\": [\"27130255\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Crystal structure of the PYCR2 apo-enzyme was determined. A disease-associated p.Gly249Val mutation lies at the dimer interface and lowers enzymatic activity. Loss of PYCR2 upregulates SHMT2, which increases cerebral glycine levels; SHMT2 knockdown partially rescued axonal beading and neurite length defects in Pycr2 knockout neurons. Loss of PYCR2 also depletes PYCR1 levels in neural lineages.\",\n      \"method\": \"Crystal structure determination; Pycr2 knockout mouse model phenotyping; in situ neurotransmitter quantification in brain; SHMT2 knockdown rescue experiments in cultured neurons; enzymatic activity assays\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure, in vitro enzymatic assay, KO mouse model, genetic epistasis via SHMT2 knockdown rescue, multiple orthogonal methods in one rigorous study\",\n      \"pmids\": [\"32330411\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PYCR2 wild-type and disease variants (R119C and R251C) were kinetically characterized; wild-type enzyme follows a sequential binding mechanism with L-P5C binding before NAD(P)H and NAD(P)+ releasing before L-Pro. Both R119C and R251C variants are catalytically impaired: R119C has 40- or 366-fold lower catalytic efficiency (with NADPH or NADH respectively), while R251C has 7- or 26-fold lower catalytic efficiency. R251C also exhibits a pronounced folding defect by thermostability and circular dichroism measurements.\",\n      \"method\": \"Steady-state kinetic measurements; thermostability assay; circular dichroism spectroscopy; in vitro enzyme assay with purified recombinant proteins\",\n      \"journal\": \"Archives of biochemistry and biophysics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified protein, kinetic mechanism established by substrate-order experiments, mutagenesis of disease variants, multiple orthogonal biophysical methods\",\n      \"pmids\": [\"33771508\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Both PYCR1 and PYCR2 localize to mitochondria in fibroblasts. Both proteins complement loss of yeast Pro3 (the P5C-to-proline reductase), confirming their activity as P5C reductases. Pycr1;Pycr2 double-mutant mice are sub-viable and worse than either single mutant, indicating largely redundant functions in proline biosynthesis.\",\n      \"method\": \"Mitochondrial localization by cell fractionation/imaging; yeast complementation assay; mouse double-mutant genetic epistasis; serum/tissue proline measurements\",\n      \"journal\": \"Genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast complementation (functional reconstitution), direct localization experiments, genetic epistasis in double-mutant mice, single lab\",\n      \"pmids\": [\"33734376\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"E4B ubiquitin E3 ligase ubiquitinates PYCR2 both in vitro and in HEK293 cells, forming K48-linked polyubiquitin chains on PYCR2 to promote its proteasomal degradation. E4B interacts with PYCR2 via its variable region.\",\n      \"method\": \"In vitro ubiquitination assay; co-immunoprecipitation in HEK293 cells; K48 linkage-specific ubiquitin chain analysis; domain mapping of E4B variable region\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro ubiquitination assay plus cell-based co-IP with linkage typing, single lab, two orthogonal methods\",\n      \"pmids\": [\"35669517\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"HLD10-associated PYCR2 mutations R119C and R251C cause formation of abnormally large mitochondria in oligodendroglial cells (FBD-102b), with increased mitochondrial fusion and decreased fission, and decreased mitochondrial activity. Cells expressing these mutants fail to undergo morphological differentiation upon induction, unlike wild-type PYCR2-expressing cells.\",\n      \"method\": \"Transfection of mutant and wild-type PYCR2 into oligodendroglial cell line; mitochondrial morphology imaging; mitochondrial fusion/fission activity assays; differentiation marker expression by western blot\",\n      \"journal\": \"Neurology international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — defined cellular phenotype with loss-of-function mutants in relevant cell type, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"36548190\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ASFV E199L protein interacts with PYCR2 (identified by co-immunoprecipitation coupled with mass spectrometry) and downregulates PYCR2 expression, resulting in autophagy activation.\",\n      \"method\": \"Co-immunoprecipitation coupled with mass spectrometry; western blot for PYCR2 expression; autophagy assays in Vero and HEK-293T cells\",\n      \"journal\": \"Virologica Sinica\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP/MS in a viral context, single lab, limited mechanistic follow-up on PYCR2 itself\",\n      \"pmids\": [\"33830435\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"c-Myc binds to the PYCR2 promoter and transcriptionally upregulates PYCR2 expression, as demonstrated by luciferase reporter assay and western blot in breast cancer cells.\",\n      \"method\": \"Luciferase reporter assay with PYCR2 promoter; western blot; chromatin binding analysis\",\n      \"journal\": \"The international journal of biochemistry & cell biology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — luciferase reporter assay for transcriptional regulation, single lab, single method for the transcriptional mechanism\",\n      \"pmids\": [\"38101533\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ALKBH5 promotes PYCR2 expression (and PYCR2-mediated proline synthesis), while PYCR2 in turn promotes ALKBH5 expression through the AMPK/mTOR pathway, forming a positive feedback loop in GBM cells. Proline supplementation rescued AMPK/mTOR activation and proneural-mesenchymal transition after PYCR2 silencing.\",\n      \"method\": \"siRNA/shRNA knockdown; western blot; proline supplementation rescue experiment; AMPK/mTOR pathway analysis\",\n      \"journal\": \"Journal of Cancer\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — pathway epistasis by knockdown and rescue, single lab, limited mechanistic resolution from abstract alone\",\n      \"pmids\": [\"37325047\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PFDN2 physically interacts with PYCR2 (co-immunoprecipitation; cytoplasmic colocalization by immunofluorescence) and stabilizes PYCR2 protein by limiting proteasome-dependent degradation, as shown by cycloheximide chase and MG132 rescue experiments. PYCR2 activity is required downstream of PFDN2 to activate Wnt/β-catenin signaling in colorectal cancer cells.\",\n      \"method\": \"Co-immunoprecipitation; immunofluorescence colocalization; cycloheximide chase assay; MG132 proteasome inhibitor rescue; TOP/FOPflash reporter assay; rescue experiments with PYCR2 re-expression\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP, protein stability assays with inhibitor rescue, pathway reporter assay, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"41656306\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PYCR2 is a mitochondria-localized enzyme that catalyzes the final step of proline biosynthesis (reduction of L-Δ1-pyrroline-5-carboxylate to L-proline via a sequential NAD(P)H-dependent mechanism); disease-associated mutations impair catalysis and/or protein folding/multimerization, and loss of PYCR2 causes neurodegeneration by upregulating SHMT2, elevating cerebral glycine, disrupting mitochondrial dynamics in oligodendrocytes, and depleting PYCR1 in neural lineages; PYCR2 protein stability is regulated by E4B-mediated K48-linked ubiquitination and by PFDN2-dependent protection from proteasomal degradation; PYCR2 also functionally collaborates with RRM2B to protect cells from oxidative stress.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PYCR2 is a mitochondrial NAD(P)H-dependent reductase that catalyzes the terminal step of proline biosynthesis, converting L-Δ1-pyrroline-5-carboxylate (L-P5C) to L-proline through an ordered sequential mechanism in which L-P5C binds before NAD(P)H and NAD(P)+ is released before L-proline [#3, #4, #5]. The enzyme is active as an oligomer—a crystal structure of the apo-enzyme places the disease-associated p.Gly249Val substitution at the dimer interface where it lowers catalytic activity, and patient missense mutations impair multimeric assembly [#2, #3]. PYCR2 functions largely redundantly with PYCR1 as a P5C reductase, both localizing to mitochondria and both complementing loss of the yeast P5C-to-proline reductase, with double-mutant mice more severely affected than either single mutant [#5]. Loss of PYCR2 causes neurodegeneration: it upregulates SHMT2 and elevates cerebral glycine, depletes PYCR1 in neural lineages, and impairs neuronal morphology, while SHMT2 knockdown partially rescues the axonal and neurite defects [#3]. Biallelic PYCR2 variants such as p.Arg119Cys and p.Arg251Cys—which retain mitochondrial targeting but are catalytically impaired and, for R251C, also misfolded—underlie hypomyelinating leukodystrophy, with mutant proteins driving abnormal mitochondrial fusion/fission balance and blocking oligodendroglial differentiation [#1, #4, #7]. PYCR2 abundance is controlled post-translationally by E4B-mediated K48-linked polyubiquitination targeting it for proteasomal degradation and by PFDN2, which binds PYCR2 and protects it from degradation [#6, #11]. Functionally, PYCR2 collaborates with RRM2B to confer resistance to oxidative stress, and its loss lowers mitochondrial membrane potential and sensitizes cells to apoptosis [#0, #1].\",\n  \"teleology\": [\n    {\n      \"year\": 2015,\n      \"claim\": \"Establishing that PYCR2 missense variants disrupt protein stability and cellular fitness rather than mislocalization linked the gene to disease through a loss-of-function mechanism.\",\n      \"evidence\": \"CRISPR-Cas9 knockout cell line with mitochondrial membrane potential and apoptosis assays, plus mutant cDNA localization imaging\",\n      \"pmids\": [\"25865492\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not resolve whether instability reflects catalytic or folding defects\", \"No structural basis for variant destabilization\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identifying impaired multimerization in patient mutants connected oligomeric assembly to PYCR2 function, framing assembly as a disease-relevant property.\",\n      \"evidence\": \"Biochemical multimerization assay of patient-derived mutations\",\n      \"pmids\": [\"27130255\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Limited methodological detail on the multimerization assay\", \"Oligomeric state not linked to catalytic output here\", \"No structural model\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Placing PYCR1/PYCR2 within RRM2B complexes and showing their joint requirement for RRM2B anti-oxidation activity revealed a role beyond proline synthesis in oxidative stress defense.\",\n      \"evidence\": \"Flag-RRM2B affinity purification with mass spectrometry plus shRNA double-knockdown under oxidative stress\",\n      \"pmids\": [\"26733354\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct physical contact between PYCR2 and RRM2B not demonstrated\", \"Mechanism of anti-oxidation contribution unresolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"A crystal structure plus a knockout mouse defined the catalytic architecture and the in vivo neurodegenerative cascade, showing loss of PYCR2 elevates glycine via SHMT2 and depletes PYCR1.\",\n      \"evidence\": \"Apo-enzyme crystal structure, Pycr2 knockout mouse phenotyping, brain neurotransmitter quantification, and SHMT2-knockdown rescue in cultured neurons\",\n      \"pmids\": [\"32330411\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking SHMT2 upregulation to PYCR2 loss not defined\", \"How PYCR1 depletion is triggered unclear\", \"No holo/substrate-bound structure\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Steady-state kinetics established the ordered catalytic mechanism and quantified how disease variants impair catalysis versus folding, distinguishing two failure modes.\",\n      \"evidence\": \"Steady-state kinetic measurements, thermostability and circular dichroism on purified recombinant wild-type and R119C/R251C proteins\",\n      \"pmids\": [\"33771508\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Substrate-bound structural states not captured\", \"Physiological cofactor preference (NADPH vs NADH) in vivo unresolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Yeast complementation and double-mutant mice confirmed PYCR2 as a bona fide P5C reductase that is largely functionally redundant with PYCR1.\",\n      \"evidence\": \"Yeast Pro3 complementation, mitochondrial localization, Pycr1;Pycr2 double-mutant mouse genetics and tissue proline measurements\",\n      \"pmids\": [\"33734376\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Non-redundant, tissue-specific roles of the two enzymes not delineated\", \"Quantitative contribution of each isozyme to proline pools unresolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"A viral interactor was found to downregulate PYCR2 and trigger autophagy, hinting at PYCR2 levels influencing autophagic signaling.\",\n      \"evidence\": \"Co-IP/MS of ASFV E199L with PYCR2, western blot and autophagy assays in Vero and HEK-293T cells\",\n      \"pmids\": [\"33830435\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single Co-IP/MS without reciprocal validation\", \"Direct causal link between PYCR2 loss and autophagy not isolated\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identifying E4B-mediated K48 polyubiquitination defined a proteasomal route controlling PYCR2 abundance.\",\n      \"evidence\": \"In vitro ubiquitination assay, co-IP in HEK293 cells, K48 linkage typing, and E4B variable-region domain mapping\",\n      \"pmids\": [\"35669517\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological contexts regulating E4B–PYCR2 not defined\", \"Deubiquitinase counterpart unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Showing that R119C/R251C drive abnormal mitochondrial fusion/fission and block oligodendroglial differentiation connected variant biochemistry to the cellular pathology of hypomyelinating leukodystrophy.\",\n      \"evidence\": \"Transfection of wild-type/mutant PYCR2 into FBD-102b oligodendroglial cells with mitochondrial morphology, fusion/fission and differentiation-marker readouts\",\n      \"pmids\": [\"36548190\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular link between reductase deficiency and fission/fusion machinery unclear\", \"Overexpression system may not reflect endogenous stoichiometry\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Transcriptional and m6A-associated regulators (c-Myc, ALKBH5) were placed upstream of PYCR2 in cancer, embedding it in proline-driven oncogenic circuits.\",\n      \"evidence\": \"Luciferase promoter reporter and western blot (c-Myc); knockdown, proline rescue and AMPK/mTOR analysis (ALKBH5 feedback loop) in breast and glioblastoma cells\",\n      \"pmids\": [\"38101533\", \"37325047\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single-method transcriptional evidence per study\", \"Direct vs indirect regulation not fully separated\", \"Generality across tumor types untested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"PFDN2 was shown to bind and stabilize PYCR2 against proteasomal turnover, with PYCR2 activity required for Wnt/β-catenin signaling, defining a stabilizing partner opposing E4B-driven degradation.\",\n      \"evidence\": \"Reciprocal co-IP, immunofluorescence colocalization, cycloheximide chase, MG132 rescue, and TOP/FOPflash reporter with PYCR2 re-expression in colorectal cancer cells\",\n      \"pmids\": [\"41656306\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How PFDN2 protects PYCR2 mechanistically (shielding vs competition with E4B) unknown\", \"Link between proline metabolism and Wnt activation not mechanistically resolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How PYCR2's enzymatic proline output is mechanistically coupled to its downstream non-metabolic phenotypes—mitochondrial dynamics, oligodendrocyte differentiation, oxidative-stress resistance and Wnt/AMPK-mTOR signaling—remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No substrate-bound structure to explain variant-specific defects\", \"Causal chain from proline depletion to mitochondrial morphology changes undefined\", \"In vivo relevance of cancer-associated regulatory loops untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016491\", \"supporting_discovery_ids\": [3, 4, 5]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [4, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [1, 5]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [11]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [3, 4, 5]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [6, 11]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"RRM2B\", \"PYCR1\", \"E4B\", \"PFDN2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}