{"gene":"PXMP4","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":2025,"finding":"MARCHF7 acts as an E3 ubiquitin ligase that binds PXMP4 and promotes its ubiquitination at lysine 20 in PEX1-deficient cells. Ubiquitinated PXMP4 acts as a recognition signal for the autophagy receptor NBR1, recruiting it to peroxisomes to drive pexophagy. TBK1 (phosphorylated at serine 172 upon PEX1 depletion, driven by ROS accumulation) phosphorylates and activates MARCHF7, establishing a TBK1-MARCHF7-PXMP4-NBR1 axis that regulates pexophagy.","method":"Functional screening, co-IP/binding assays, ubiquitination assays, site-directed mutagenesis (K20 mutant), knockdown/reconstitution experiments, pexophagic flux assays","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal binding, ubiquitination site mutagenesis (K20), reconstitution with ubiquitination-defective mutant, multiple orthogonal methods in a single focused study","pmids":["41267209"],"is_preprint":false},{"year":2022,"finding":"Pxmp4 knockout mice (generated by CRISPR/Cas9) are viable and fertile with no changes in peroxisome numbers or morphology, and no differences in plasma VLCFAs, bile acids, or bile acid intermediates. However, loss of Pxmp4 decreased hepatic levels of alkyldiacylglycerol ether lipids (particularly those containing polyunsaturated fatty acids), suggesting a role in ether lipid metabolism. Elevated phytanic/pristanic acid levels in Pxmp4-/- mice also pointed toward impaired peroxisomal α-oxidation capacity.","method":"CRISPR/Cas9 knockout mouse model, lipidomic analysis, plasma metabolite measurements (VLCFAs, bile acids), phytol-enriched diet challenge","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO mouse with multiple defined metabolic readouts and lipidomic analysis across multiple conditions","pmids":["35169201"],"is_preprint":false},{"year":2024,"finding":"PXMP4 knockdown/overexpression in gastric cancer cells modulates proliferation, invasion, and migration, and regulates epithelial-mesenchymal transition (EMT) through activation of the PI3K/AKT signaling pathway. The PI3K/AKT inhibitor LY294002 inhibited PI3K/AKT-related proteins but did not affect PXMP4 expression, placing PXMP4 upstream of PI3K/AKT in this pathway.","method":"siRNA knockdown and overexpression in gastric cancer cell lines, PI3K/AKT inhibitor (LY294002) treatment, proliferation/invasion/migration assays, Western blot for EMT and pathway markers","journal":"Molecular biology reports","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — KD/OE with cellular phenotype and pathway inhibitor epistasis, single lab, no in vitro reconstitution","pmids":["38401002"],"is_preprint":false},{"year":2024,"finding":"PEX19 binds to the peroxisomal membrane protein PMP24 (PXMP4), and the interaction between influenza A virus M2 protein and PEX19 disrupts this PEX19-PMP24 interaction, implicating PXMP4 as part of the PEX19-dependent peroxisomal membrane insertion machinery.","method":"Co-IP/binding assays between PEX19, M2 protein, PEX14, and PMP24; PEX19 knockdown; viral titer measurements","journal":"Viruses","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — single Co-IP showing disrupted PEX19-PMP24 interaction; indirect evidence, single lab","pmids":["39205283"],"is_preprint":false},{"year":2004,"finding":"Transient transfection of PMP24 (PXMP4) into LNCaP(CS) and PC-3 prostate cancer cells (which lack endogenous PMP24 expression due to CpG island hypermethylation) induced a significant reduction in cell growth and soft-agar colony formation, suggesting an anti-tumor/anti-proliferative function for the gene product.","method":"Transient transfection, cell growth assay, soft-agar colony formation assay","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — overexpression with two functional readouts (growth and anchorage-independent growth), single lab, no molecular mechanism identified","pmids":["14712230"],"is_preprint":false},{"year":2010,"finding":"Methylation of a single intronic CpG dinucleotide (the first CpG of a hypersensitive site in intron 1) of PXMP4 is sufficient to disrupt DNA-protein interactions at that site and suppress gene expression, as demonstrated by gel shift assays and transfection of a methylated oligonucleotide corresponding to that site.","method":"Limited demethylation (low-dose 5-aza-dC), gel shift assay, methylated oligonucleotide transfection, bisulfite sequencing, in situ hybridization","journal":"The Prostate","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — site-specific methylation transfection combined with gel shift and bisulfite sequencing, single lab, multiple orthogonal methods","pmids":["20054818"],"is_preprint":false},{"year":2016,"finding":"Sequence analysis classified PXMP4 (Pmp24) as a member of the Tim17 protein family, which broadly functions in membrane transport/translocation, suggesting PXMP4 may function as a channel or transporter in the peroxisomal membrane.","method":"Comprehensive sequence/phylogenetic analysis of 5631 proteomes","journal":"Biology direct","confidence":"Low","confidence_rationale":"Tier 4 / Weak — computational sequence analysis only, no experimental functional validation of PXMP4 specifically","pmids":["27760563"],"is_preprint":false},{"year":2008,"finding":"Microarray expression analysis of a NOD.Nkrp1b.Nkt2bb congenic mouse strain identified Pxmp4 as one of 19 highly differentially expressed candidate genes within the Nkt2 region. The paper notes that the only known binding partner of PXMP4 is PEX19 (an intracellular chaperone for peroxisomal membrane insertion), raising the possibility that peroxisomes modulate glycolipid availability for CD1d presentation to NKT cells.","method":"Congenic mouse strain generation, microarray expression analysis","journal":"Journal of immunology","confidence":"Low","confidence_rationale":"Tier 4 / Weak — expression-based candidate gene identification; the PEX19 binding partner relationship is cited as prior knowledge, not directly demonstrated in this paper","pmids":["18714012"],"is_preprint":false}],"current_model":"PXMP4 (PMP24) is a peroxisomal membrane protein that participates in pexophagy as a ubiquitination substrate: the E3 ligase MARCHF7 (activated downstream of TBK1 via ROS/PEX1 depletion) ubiquitinates PXMP4 at lysine 20, generating a signal recognized by the autophagy receptor NBR1 to drive selective peroxisome degradation; it also interacts with PEX19 (the peroxisomal membrane insertion chaperone), has a role in hepatic ether lipid metabolism (revealed by knockout mice), and its expression is epigenetically silenced by methylation of a single intronic CpG dinucleotide in cancer contexts."},"narrative":{"mechanistic_narrative":"PXMP4 (PMP24) is a peroxisomal membrane protein that functions both as a metabolic contributor to peroxisomal lipid processing and as a regulatory node in selective peroxisome autophagy [PMID:41267209, PMID:35169201]. In pexophagy, PXMP4 serves as a ubiquitination substrate within a defined stress-responsive cascade: upon PEX1 depletion and ROS accumulation, TBK1 is phosphorylated at serine 172 and activates the E3 ubiquitin ligase MARCHF7, which binds PXMP4 and ubiquitinates it at lysine 20; the ubiquitin mark recruits the autophagy receptor NBR1 to peroxisomes to drive their selective degradation [PMID:41267209]. Loss of Pxmp4 in mice is compatible with normal peroxisome number and morphology but reduces hepatic alkyldiacylglycerol ether lipids and elevates phytanic/pristanic acid levels, indicating a role in ether lipid metabolism and peroxisomal α-oxidation capacity [PMID:35169201]. PXMP4 is a binding partner of PEX19, the chaperone for peroxisomal membrane protein insertion, placing it within the PEX19-dependent membrane assembly machinery [PMID:39205283]. In cancer contexts, PXMP4 expression is silenced by methylation of a single intronic CpG dinucleotide that disrupts DNA-protein binding at an intron 1 hypersensitive site [PMID:20054818], and restored PXMP4 expression suppresses prostate cancer cell growth and anchorage-independent colony formation [PMID:14712230]; in gastric cancer cells it modulates proliferation, invasion, and EMT upstream of PI3K/AKT signaling [PMID:38401002].","teleology":[{"year":2004,"claim":"Established that PXMP4 has anti-proliferative activity, addressing whether the gene product had any functional consequence when re-expressed in cells that had silenced it.","evidence":"Transient transfection of PMP24 into prostate cancer cells lacking endogenous expression, with growth and soft-agar colony assays","pmids":["14712230"],"confidence":"Medium","gaps":["No molecular mechanism for the growth suppression identified","Overexpression in two cell lines only, single lab","Does not link the phenotype to peroxisomal function"]},{"year":2008,"claim":"Flagged PXMP4 as a differentially expressed candidate within an NKT-cell-linked locus, raising the question of whether peroxisomal lipid handling influences glycolipid antigen presentation.","evidence":"Microarray expression analysis of a NOD congenic mouse strain","pmids":["18714012"],"confidence":"Low","gaps":["Expression correlation only, no functional test","PEX19 binding partner cited as prior knowledge, not demonstrated here","No causal link between PXMP4 and NKT phenotype"]},{"year":2010,"claim":"Defined the mechanism of PXMP4 silencing in cancer, answering how loss of expression is established at the DNA level.","evidence":"Site-specific methylated-oligonucleotide transfection, gel shift, bisulfite sequencing in prostate tissue","pmids":["20054818"],"confidence":"Medium","gaps":["Identity of the DNA-binding factor displaced by methylation not determined","Single intronic CpG mechanism not validated in vivo across tumors","Does not address PXMP4 protein function"]},{"year":2016,"claim":"Proposed by sequence homology that PXMP4 belongs to the Tim17 translocase family, framing a hypothesis that it acts as a peroxisomal membrane channel/transporter.","evidence":"Comprehensive phylogenetic analysis across 5631 proteomes","pmids":["27760563"],"confidence":"Low","gaps":["No experimental transport activity demonstrated for PXMP4","Substrate(s) unknown","Family assignment computational only"]},{"year":2022,"claim":"Determined the physiological consequence of PXMP4 loss in vivo, resolving whether the protein is required for peroxisome integrity versus specific metabolic functions.","evidence":"CRISPR/Cas9 Pxmp4 knockout mouse with lipidomics and plasma metabolite profiling under dietary challenge","pmids":["35169201"],"confidence":"High","gaps":["Molecular basis linking PXMP4 to ether lipid and α-oxidation changes not defined","No direct transport/enzymatic substrate identified","Hepatic focus; other tissues not characterized"]},{"year":2024,"claim":"Provided direct evidence that PXMP4 interacts with PEX19, anchoring it within the peroxisomal membrane insertion machinery and showing the interaction is virally targetable.","evidence":"Co-IP between PEX19, PMP24, PEX14 and influenza M2 protein, with PEX19 knockdown and viral titer assays","pmids":["39205283"],"confidence":"Medium","gaps":["Single Co-IP showing disruption; no reciprocal validation of functional consequence for PXMP4 insertion","Does not map the PEX19-binding region of PXMP4","Single lab"]},{"year":2024,"claim":"Placed PXMP4 upstream of PI3K/AKT signaling in gastric cancer, connecting its expression to a defined oncogenic pathway and EMT.","evidence":"siRNA knockdown/overexpression with LY294002 epistasis and EMT/pathway Western blots in gastric cancer lines","pmids":["38401002"],"confidence":"Medium","gaps":["Mechanism by which PXMP4 activates PI3K/AKT not defined","Single lab, no in vitro reconstitution","Relationship to PXMP4's peroxisomal function unclear"]},{"year":2025,"claim":"Resolved a specific regulatory role for PXMP4 as the ubiquitination substrate that licenses pexophagy, defining a complete TBK1-MARCHF7-PXMP4-NBR1 signaling axis.","evidence":"Functional screening, reciprocal binding and ubiquitination assays, K20 site mutagenesis, reconstitution, and pexophagic flux assays in PEX1-deficient cells","pmids":["41267209"],"confidence":"High","gaps":["How widely the axis operates beyond PEX1-deficient/ROS contexts unknown","Structural basis of MARCHF7-PXMP4 recognition not resolved","Relationship between PXMP4 ubiquitination and its metabolic role undefined"]},{"year":null,"claim":"Whether PXMP4's proposed channel/transporter activity, its ether-lipid metabolic role, and its function as a pexophagy substrate represent one integrated biochemical activity or distinct roles remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No demonstrated transport substrate for PXMP4","No structural model of the protein","Link between metabolic phenotypes and the ubiquitination/autophagy axis not established"]}],"mechanism_profile":{"molecular_activity":[],"localization":[{"term_id":"GO:0005777","term_label":"peroxisome","supporting_discovery_ids":[0,1,3]}],"pathway":[{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[0]}],"complexes":[],"partners":["MARCHF7","NBR1","PEX19"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9Y6I8","full_name":"Peroxisomal membrane protein 4","aliases":["24 kDa peroxisomal intrinsic membrane protein"],"length_aa":212,"mass_kda":24.3,"function":"","subcellular_location":"Peroxisome membrane","url":"https://www.uniprot.org/uniprotkb/Q9Y6I8/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PXMP4","classification":"Not Classified","n_dependent_lines":3,"n_total_lines":1208,"dependency_fraction":0.0024834437086092716},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/PXMP4","total_profiled":1310},"omim":[{"mim_id":"616397","title":"PEROXISOMAL MEMBRANE PROTEIN 4; PXMP4","url":"https://www.omim.org/entry/616397"},{"mim_id":"602750","title":"D-DOPACHROME TAUTOMERASE; DDT","url":"https://www.omim.org/entry/602750"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Peroxisomes","reliability":"Approved"},{"location":"Nucleoli fibrillar center","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PXMP4"},"hgnc":{"alias_symbol":["PMP24"],"prev_symbol":[]},"alphafold":{"accession":"Q9Y6I8","domains":[{"cath_id":"-","chopping":"18-117","consensus_level":"medium","plddt":94.1928,"start":18,"end":117},{"cath_id":"-","chopping":"153-206","consensus_level":"medium","plddt":91.7693,"start":153,"end":206},{"cath_id":"1.20.5","chopping":"118-150","consensus_level":"medium","plddt":94.1788,"start":118,"end":150}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y6I8","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y6I8-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y6I8-F1-predicted_aligned_error_v6.png","plddt_mean":91.81},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PXMP4","jax_strain_url":"https://www.jax.org/strain/search?query=PXMP4"},"sequence":{"accession":"Q9Y6I8","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9Y6I8.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9Y6I8/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y6I8"}},"corpus_meta":[{"pmid":"19710929","id":"PMC_19710929","title":"Comparative analysis of gene regulation by the transcription factor PPARalpha between mouse and human.","date":"2009","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/19710929","citation_count":265,"is_preprint":false},{"pmid":"7721939","id":"PMC_7721939","title":"Pmp27 promotes peroxisomal proliferation.","date":"1995","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/7721939","citation_count":179,"is_preprint":false},{"pmid":"27760563","id":"PMC_27760563","title":"Evolution of the Tim17 protein family.","date":"2016","source":"Biology direct","url":"https://pubmed.ncbi.nlm.nih.gov/27760563","citation_count":56,"is_preprint":false},{"pmid":"20054818","id":"PMC_20054818","title":"Methylation of a single intronic CpG mediates expression silencing of the PMP24 gene in prostate cancer.","date":"2010","source":"The Prostate","url":"https://pubmed.ncbi.nlm.nih.gov/20054818","citation_count":53,"is_preprint":false},{"pmid":"24358884","id":"PMC_24358884","title":"Proteins and lipids of glycosomal membranes from Leishmania tarentolae and Trypanosoma brucei.","date":"2013","source":"F1000Research","url":"https://pubmed.ncbi.nlm.nih.gov/24358884","citation_count":37,"is_preprint":false},{"pmid":"17041235","id":"PMC_17041235","title":"MMASS: an optimized array-based method for assessing CpG island methylation.","date":"2006","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/17041235","citation_count":34,"is_preprint":false},{"pmid":"14712230","id":"PMC_14712230","title":"PMP24, a gene identified by MSRF, undergoes DNA hypermethylation-associated gene silencing during cancer progression in an LNCaP model.","date":"2004","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/14712230","citation_count":29,"is_preprint":false},{"pmid":"18714012","id":"PMC_18714012","title":"Congenic analysis of the NKT cell control gene Nkt2 implicates the peroxisomal protein Pxmp4.","date":"2008","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/18714012","citation_count":27,"is_preprint":false},{"pmid":"25271210","id":"PMC_25271210","title":"Identification of genes whose expression is altered by obesity throughout the arterial tree.","date":"2014","source":"Physiological genomics","url":"https://pubmed.ncbi.nlm.nih.gov/25271210","citation_count":23,"is_preprint":false},{"pmid":"32265992","id":"PMC_32265992","title":"HSD17B4, ACAA1, and PXMP4 in Peroxisome Pathway Are Down-Regulated and Have Clinical Significance in Non-small Cell Lung Cancer.","date":"2020","source":"Frontiers in genetics","url":"https://pubmed.ncbi.nlm.nih.gov/32265992","citation_count":19,"is_preprint":false},{"pmid":"33387576","id":"PMC_33387576","title":"Identification of four methylation-driven genes as candidate biomarkers for monitoring single-walled carbon nanotube-induced malignant transformation of the lung.","date":"2020","source":"Toxicology and applied pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/33387576","citation_count":14,"is_preprint":false},{"pmid":"35169201","id":"PMC_35169201","title":"Mice with a deficiency in Peroxisomal Membrane Protein 4 (PXMP4) display mild changes in hepatic lipid metabolism.","date":"2022","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/35169201","citation_count":13,"is_preprint":false},{"pmid":"33791307","id":"PMC_33791307","title":"Study on the Reparative Effect of PEGylated Growth Hormone on Ovarian Parameters and Mitochondrial Function of Oocytes From Rats With Premature Ovarian Insufficiency.","date":"2021","source":"Frontiers in cell and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/33791307","citation_count":12,"is_preprint":false},{"pmid":"34867906","id":"PMC_34867906","title":"Copper Tolerance Mechanism of the Novel Marine Multi-Stress Tolerant Yeast Meyerozyma guilliermondii GXDK6 as Revealed by Integrated Omics Analysis.","date":"2021","source":"Frontiers in microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/34867906","citation_count":11,"is_preprint":false},{"pmid":"39205283","id":"PMC_39205283","title":"The M2 Protein of the Influenza A Virus Interacts with PEX19 to Facilitate Virus Replication by Disrupting the Function of Peroxisome.","date":"2024","source":"Viruses","url":"https://pubmed.ncbi.nlm.nih.gov/39205283","citation_count":4,"is_preprint":false},{"pmid":"38401002","id":"PMC_38401002","title":"PXMP4 promotes gastric cancer cell epithelial-mesenchymal transition via the PI3K/AKT signaling pathway.","date":"2024","source":"Molecular biology reports","url":"https://pubmed.ncbi.nlm.nih.gov/38401002","citation_count":2,"is_preprint":false},{"pmid":"37560637","id":"PMC_37560637","title":"Lnc-PXMP4-2-4 alleviates myocardial cell damage by activating the JAK2/STAT3 signaling pathway.","date":"2023","source":"Heliyon","url":"https://pubmed.ncbi.nlm.nih.gov/37560637","citation_count":2,"is_preprint":false},{"pmid":"35694189","id":"PMC_35694189","title":"Characterizing the KRAS G12C mutation in metastatic colorectal cancer: a population-based cohort and assessment of expression differences in The Cancer Genome Atlas.","date":"2022","source":"Therapeutic advances in medical oncology","url":"https://pubmed.ncbi.nlm.nih.gov/35694189","citation_count":2,"is_preprint":false},{"pmid":"35464075","id":"PMC_35464075","title":"Alteration of Ileal lncRNAs After Duodenal-Jejunal Bypass Is Associated With Regulation of Lipid and Amino Acid Metabolism.","date":"2022","source":"Frontiers in physiology","url":"https://pubmed.ncbi.nlm.nih.gov/35464075","citation_count":2,"is_preprint":false},{"pmid":"41267209","id":"PMC_41267209","title":"Regulation of pexophagy by a novel TBK1-MARCHF7-PXMP4-NBR1 axis in PEX1-depleted HeLa cells.","date":"2025","source":"Autophagy","url":"https://pubmed.ncbi.nlm.nih.gov/41267209","citation_count":1,"is_preprint":false},{"pmid":"38415547","id":"PMC_38415547","title":"High Expression of PXMP4 in Hepatocellular Carcinoma Tissues.","date":"2024","source":"Asian Pacific journal of cancer prevention : APJCP","url":"https://pubmed.ncbi.nlm.nih.gov/38415547","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":13399,"output_tokens":2241,"usd":0.036906,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9231,"output_tokens":2948,"usd":0.059928,"stage2_stop_reason":"end_turn"},"total_usd":0.096834,"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\": 2025,\n      \"finding\": \"MARCHF7 acts as an E3 ubiquitin ligase that binds PXMP4 and promotes its ubiquitination at lysine 20 in PEX1-deficient cells. Ubiquitinated PXMP4 acts as a recognition signal for the autophagy receptor NBR1, recruiting it to peroxisomes to drive pexophagy. TBK1 (phosphorylated at serine 172 upon PEX1 depletion, driven by ROS accumulation) phosphorylates and activates MARCHF7, establishing a TBK1-MARCHF7-PXMP4-NBR1 axis that regulates pexophagy.\",\n      \"method\": \"Functional screening, co-IP/binding assays, ubiquitination assays, site-directed mutagenesis (K20 mutant), knockdown/reconstitution experiments, pexophagic flux assays\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal binding, ubiquitination site mutagenesis (K20), reconstitution with ubiquitination-defective mutant, multiple orthogonal methods in a single focused study\",\n      \"pmids\": [\"41267209\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Pxmp4 knockout mice (generated by CRISPR/Cas9) are viable and fertile with no changes in peroxisome numbers or morphology, and no differences in plasma VLCFAs, bile acids, or bile acid intermediates. However, loss of Pxmp4 decreased hepatic levels of alkyldiacylglycerol ether lipids (particularly those containing polyunsaturated fatty acids), suggesting a role in ether lipid metabolism. Elevated phytanic/pristanic acid levels in Pxmp4-/- mice also pointed toward impaired peroxisomal α-oxidation capacity.\",\n      \"method\": \"CRISPR/Cas9 knockout mouse model, lipidomic analysis, plasma metabolite measurements (VLCFAs, bile acids), phytol-enriched diet challenge\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO mouse with multiple defined metabolic readouts and lipidomic analysis across multiple conditions\",\n      \"pmids\": [\"35169201\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PXMP4 knockdown/overexpression in gastric cancer cells modulates proliferation, invasion, and migration, and regulates epithelial-mesenchymal transition (EMT) through activation of the PI3K/AKT signaling pathway. The PI3K/AKT inhibitor LY294002 inhibited PI3K/AKT-related proteins but did not affect PXMP4 expression, placing PXMP4 upstream of PI3K/AKT in this pathway.\",\n      \"method\": \"siRNA knockdown and overexpression in gastric cancer cell lines, PI3K/AKT inhibitor (LY294002) treatment, proliferation/invasion/migration assays, Western blot for EMT and pathway markers\",\n      \"journal\": \"Molecular biology reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — KD/OE with cellular phenotype and pathway inhibitor epistasis, single lab, no in vitro reconstitution\",\n      \"pmids\": [\"38401002\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PEX19 binds to the peroxisomal membrane protein PMP24 (PXMP4), and the interaction between influenza A virus M2 protein and PEX19 disrupts this PEX19-PMP24 interaction, implicating PXMP4 as part of the PEX19-dependent peroxisomal membrane insertion machinery.\",\n      \"method\": \"Co-IP/binding assays between PEX19, M2 protein, PEX14, and PMP24; PEX19 knockdown; viral titer measurements\",\n      \"journal\": \"Viruses\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP showing disrupted PEX19-PMP24 interaction; indirect evidence, single lab\",\n      \"pmids\": [\"39205283\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Transient transfection of PMP24 (PXMP4) into LNCaP(CS) and PC-3 prostate cancer cells (which lack endogenous PMP24 expression due to CpG island hypermethylation) induced a significant reduction in cell growth and soft-agar colony formation, suggesting an anti-tumor/anti-proliferative function for the gene product.\",\n      \"method\": \"Transient transfection, cell growth assay, soft-agar colony formation assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — overexpression with two functional readouts (growth and anchorage-independent growth), single lab, no molecular mechanism identified\",\n      \"pmids\": [\"14712230\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Methylation of a single intronic CpG dinucleotide (the first CpG of a hypersensitive site in intron 1) of PXMP4 is sufficient to disrupt DNA-protein interactions at that site and suppress gene expression, as demonstrated by gel shift assays and transfection of a methylated oligonucleotide corresponding to that site.\",\n      \"method\": \"Limited demethylation (low-dose 5-aza-dC), gel shift assay, methylated oligonucleotide transfection, bisulfite sequencing, in situ hybridization\",\n      \"journal\": \"The Prostate\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — site-specific methylation transfection combined with gel shift and bisulfite sequencing, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"20054818\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Sequence analysis classified PXMP4 (Pmp24) as a member of the Tim17 protein family, which broadly functions in membrane transport/translocation, suggesting PXMP4 may function as a channel or transporter in the peroxisomal membrane.\",\n      \"method\": \"Comprehensive sequence/phylogenetic analysis of 5631 proteomes\",\n      \"journal\": \"Biology direct\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — computational sequence analysis only, no experimental functional validation of PXMP4 specifically\",\n      \"pmids\": [\"27760563\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Microarray expression analysis of a NOD.Nkrp1b.Nkt2bb congenic mouse strain identified Pxmp4 as one of 19 highly differentially expressed candidate genes within the Nkt2 region. The paper notes that the only known binding partner of PXMP4 is PEX19 (an intracellular chaperone for peroxisomal membrane insertion), raising the possibility that peroxisomes modulate glycolipid availability for CD1d presentation to NKT cells.\",\n      \"method\": \"Congenic mouse strain generation, microarray expression analysis\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — expression-based candidate gene identification; the PEX19 binding partner relationship is cited as prior knowledge, not directly demonstrated in this paper\",\n      \"pmids\": [\"18714012\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PXMP4 (PMP24) is a peroxisomal membrane protein that participates in pexophagy as a ubiquitination substrate: the E3 ligase MARCHF7 (activated downstream of TBK1 via ROS/PEX1 depletion) ubiquitinates PXMP4 at lysine 20, generating a signal recognized by the autophagy receptor NBR1 to drive selective peroxisome degradation; it also interacts with PEX19 (the peroxisomal membrane insertion chaperone), has a role in hepatic ether lipid metabolism (revealed by knockout mice), and its expression is epigenetically silenced by methylation of a single intronic CpG dinucleotide in cancer contexts.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PXMP4 (PMP24) is a peroxisomal membrane protein that functions both as a metabolic contributor to peroxisomal lipid processing and as a regulatory node in selective peroxisome autophagy [#0, #1]. In pexophagy, PXMP4 serves as a ubiquitination substrate within a defined stress-responsive cascade: upon PEX1 depletion and ROS accumulation, TBK1 is phosphorylated at serine 172 and activates the E3 ubiquitin ligase MARCHF7, which binds PXMP4 and ubiquitinates it at lysine 20; the ubiquitin mark recruits the autophagy receptor NBR1 to peroxisomes to drive their selective degradation [#0]. Loss of Pxmp4 in mice is compatible with normal peroxisome number and morphology but reduces hepatic alkyldiacylglycerol ether lipids and elevates phytanic/pristanic acid levels, indicating a role in ether lipid metabolism and peroxisomal \\u03b1-oxidation capacity [#1]. PXMP4 is a binding partner of PEX19, the chaperone for peroxisomal membrane protein insertion, placing it within the PEX19-dependent membrane assembly machinery [#3]. In cancer contexts, PXMP4 expression is silenced by methylation of a single intronic CpG dinucleotide that disrupts DNA-protein binding at an intron 1 hypersensitive site [#5], and restored PXMP4 expression suppresses prostate cancer cell growth and anchorage-independent colony formation [#4]; in gastric cancer cells it modulates proliferation, invasion, and EMT upstream of PI3K/AKT signaling [#2].\"\n  ,\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Established that PXMP4 has anti-proliferative activity, addressing whether the gene product had any functional consequence when re-expressed in cells that had silenced it.\",\n      \"evidence\": \"Transient transfection of PMP24 into prostate cancer cells lacking endogenous expression, with growth and soft-agar colony assays\",\n      \"pmids\": [\"14712230\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"No molecular mechanism for the growth suppression identified\", \"Overexpression in two cell lines only, single lab\", \"Does not link the phenotype to peroxisomal function\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Flagged PXMP4 as a differentially expressed candidate within an NKT-cell-linked locus, raising the question of whether peroxisomal lipid handling influences glycolipid antigen presentation.\",\n      \"evidence\": \"Microarray expression analysis of a NOD congenic mouse strain\",\n      \"pmids\": [\"18714012\"],\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Expression correlation only, no functional test\", \"PEX19 binding partner cited as prior knowledge, not demonstrated here\", \"No causal link between PXMP4 and NKT phenotype\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Defined the mechanism of PXMP4 silencing in cancer, answering how loss of expression is established at the DNA level.\",\n      \"evidence\": \"Site-specific methylated-oligonucleotide transfection, gel shift, bisulfite sequencing in prostate tissue\",\n      \"pmids\": [\"20054818\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Identity of the DNA-binding factor displaced by methylation not determined\", \"Single intronic CpG mechanism not validated in vivo across tumors\", \"Does not address PXMP4 protein function\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Proposed by sequence homology that PXMP4 belongs to the Tim17 translocase family, framing a hypothesis that it acts as a peroxisomal membrane channel/transporter.\",\n      \"evidence\": \"Comprehensive phylogenetic analysis across 5631 proteomes\",\n      \"pmids\": [\"27760563\"],\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"No experimental transport activity demonstrated for PXMP4\", \"Substrate(s) unknown\", \"Family assignment computational only\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Determined the physiological consequence of PXMP4 loss in vivo, resolving whether the protein is required for peroxisome integrity versus specific metabolic functions.\",\n      \"evidence\": \"CRISPR/Cas9 Pxmp4 knockout mouse with lipidomics and plasma metabolite profiling under dietary challenge\",\n      \"pmids\": [\"35169201\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Molecular basis linking PXMP4 to ether lipid and \\u03b1-oxidation changes not defined\", \"No direct transport/enzymatic substrate identified\", \"Hepatic focus; other tissues not characterized\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Provided direct evidence that PXMP4 interacts with PEX19, anchoring it within the peroxisomal membrane insertion machinery and showing the interaction is virally targetable.\",\n      \"evidence\": \"Co-IP between PEX19, PMP24, PEX14 and influenza M2 protein, with PEX19 knockdown and viral titer assays\",\n      \"pmids\": [\"39205283\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Single Co-IP showing disruption; no reciprocal validation of functional consequence for PXMP4 insertion\", \"Does not map the PEX19-binding region of PXMP4\", \"Single lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Placed PXMP4 upstream of PI3K/AKT signaling in gastric cancer, connecting its expression to a defined oncogenic pathway and EMT.\",\n      \"evidence\": \"siRNA knockdown/overexpression with LY294002 epistasis and EMT/pathway Western blots in gastric cancer lines\",\n      \"pmids\": [\"38401002\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Mechanism by which PXMP4 activates PI3K/AKT not defined\", \"Single lab, no in vitro reconstitution\", \"Relationship to PXMP4's peroxisomal function unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Resolved a specific regulatory role for PXMP4 as the ubiquitination substrate that licenses pexophagy, defining a complete TBK1-MARCHF7-PXMP4-NBR1 signaling axis.\",\n      \"evidence\": \"Functional screening, reciprocal binding and ubiquitination assays, K20 site mutagenesis, reconstitution, and pexophagic flux assays in PEX1-deficient cells\",\n      \"pmids\": [\"41267209\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"How widely the axis operates beyond PEX1-deficient/ROS contexts unknown\", \"Structural basis of MARCHF7-PXMP4 recognition not resolved\", \"Relationship between PXMP4 ubiquitination and its metabolic role undefined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Whether PXMP4's proposed channel/transporter activity, its ether-lipid metabolic role, and its function as a pexophagy substrate represent one integrated biochemical activity or distinct roles remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"No demonstrated transport substrate for PXMP4\", \"No structural model of the protein\", \"Link between metabolic phenotypes and the ubiquitination/autophagy axis not established\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [],\n    \"localization\": [\n      {\"term_id\": \"GO:0005777\", \"supporting_discovery_ids\": [0, 1, 3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"MARCHF7\", \"NBR1\", \"PEX19\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"faith_supported":5,"faith_total":5,"faith_pct":100.0}}