{"gene":"APOO","run_date":"2026-04-28T17:12:37","timeline":{"discoveries":[{"year":2006,"finding":"APOO (ApoO) is a novel apolipoprotein that colocalizes with perilipins at lipid droplets, belongs to the proteoglycan family (contains a chondroitin sulfate chain), requires microsomal triglyceride transfer protein (MTP) activity for secretion, and promotes cholesterol efflux from macrophages.","method":"Confocal immunofluorescence colocalization with perilipins; chondroitinase ABC deglycosylation; p-nitrophenyl-β-d-xyloside treatment; MTP inhibitor (CP-346086) treatment; cholesterol efflux assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal biochemical methods in a single lab establishing molecular properties and a functional activity","pmids":["16956892"],"is_preprint":false},{"year":2015,"finding":"The non-glycosylated 22 kDa isoform of MIC26 (APOO) spans the mitochondrial inner membrane and physically interacts with MICOS subunits MIC60, MIC27, and MIC10; its depletion reduces crista junction number and mitochondrial oxygen consumption, while overexpression induces mitochondrial fragmentation, ROS formation, and impaired respiration. MIC26 and MIC27 regulate each other's levels antagonistically, and both are positively correlated with MIC10 and tafazzin levels.","method":"Co-immunoprecipitation; miRNA-mediated knockdown; overexpression; electron microscopy of cristae ultrastructure; mitochondrial respiration assay; ROS measurement; confocal microscopy of mitochondrial network","journal":"Biochimica et biophysica acta","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP with multiple MICOS subunits, loss-of-function with defined ultrastructural and functional phenotypes, replicated across multiple experiments","pmids":["25764979"],"is_preprint":false},{"year":2015,"finding":"miRNA-mediated depletion of MIC26 increases MIC27 protein levels and decreases MIC10 levels; a miRNA-resistant form of MIC26 confirmed specificity. Depletion selectively reduces the 22 kDa mitochondrial isoform but not the 55 kDa secreted isoform; overexpression of myc-tagged MIC26 alters cristae morphology, producing swollen and vesicular cristae structures.","method":"miRNA knockdown with rescue; Western blot; electron microscopy","journal":"Data in brief","confidence":"Medium","confidence_rationale":"Tier 2 — rescue experiment confirms specificity; single lab","pmids":["26217776"],"is_preprint":false},{"year":2018,"finding":"MIC26 (APOO) exerts a destabilizing effect on MIC10 oligomers, functioning antagonistically to MIC27, which stabilizes MIC10 oligomers; cardiolipin also stabilizes MIC10 oligomers within the MICOS complex.","method":"Blue-native PAGE; biochemical analysis of Mic10 oligomeric state in yeast and mammalian cells with Mic26/Mic27 deletion","journal":"Journal of molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 — genetic deletion with defined biochemical phenotype; single lab","pmids":["29733859"],"is_preprint":false},{"year":2020,"finding":"MIC26 and MIC27 are cooperatively required for integrity of respiratory chain supercomplexes and F1Fo-ATP synthase; double knockout (DKO) of MIC26 and MIC27 causes onion-like cristae with loss of crista junctions, reduces cardiolipin levels, and impairs integration of F1 subunits into monomeric F1Fo-ATP synthase. Overexpression of cardiolipin synthase in DKO rescues respiratory chain supercomplex stability. MIC26 and MIC27 assemble late into the MICOS complex and are dispensable for stability of remaining MICOS subunits.","method":"Single and double KO human cells; complexome profiling; STED nanoscopy; blue-native gel electrophoresis; cardiolipin synthase overexpression rescue","journal":"Life science alliance","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal methods (complexome profiling, super-resolution microscopy, BN-PAGE), genetic rescue, single lab with rigorous controls","pmids":["32788226"],"is_preprint":false},{"year":2020,"finding":"A missense mutation (I117T) in APOO/MIC26 impairs protein import and insertion into the inner mitochondrial membrane, causing altered MICOS assembly and crista junction disruption. Corresponding mutations in yeast and Drosophila MIC26 orthologs also cause mitochondrial structural and functional deficiencies, confirming a conserved role in MICOS assembly.","method":"Patient fibroblast studies; whole exome sequencing; protein import assays; electron microscopy; yeast and Drosophila knockout models","journal":"Journal of medical genetics","confidence":"High","confidence_rationale":"Tier 2 — pathogenic variant with mechanistic follow-up in human cells and two model organisms, multiple orthogonal methods","pmids":["32439808"],"is_preprint":false},{"year":2023,"finding":"MIC26 (APOO) and MIC27 are exclusively localized to mitochondria as 22 kDa and 30 kDa proteins respectively; the previously reported 55 kDa glycosylated secreted MIC26 isoform is nonspecific, as confirmed by KO of MIC26 in four human cell lines using four different antibodies, GFP/myc-tagged overexpression, glycosylation site mutagenesis, and mass spectrometry.","method":"CRISPR KO in four human cell lines; multiple anti-MIC26 antibodies; tagged protein overexpression; glycosylation site mutagenesis; mass spectrometry of gel band","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal approaches in multiple cell lines definitively resolve isoform identity","pmids":["37279200"],"is_preprint":false},{"year":2023,"finding":"A nonsense mutation (E178*) in APOO/MIC26 causing loss of 20 C-terminal amino acids produces a highly unstable, degradation-prone protein; the residual mutant MIC26 correctly localizes to mitochondria and interacts physically with other MICOS subunits, but MIC26 KO cells expressing this mutant show perturbed cristae architecture and fragmented mitochondria resembling full KO.","method":"Patient exome sequencing; protein stability assays; mitochondrial localization by immunofluorescence; co-immunoprecipitation; electron microscopy of cristae in KO rescue cells","journal":"Clinical genetics","confidence":"Medium","confidence_rationale":"Tier 2 — multiple methods in patient-derived and engineered cells; single lab","pmids":["37649161"],"is_preprint":false},{"year":2023,"finding":"Macrophage-specific deletion of MIC26 (APOO) reduces mitochondrial OPA1 protein, increases mitochondrial fission, decreases mitochondrial membrane potential, and promotes efferocytosis; OPA1 overexpression abolishes the increased efferocytosis caused by MIC26 deficiency, placing MIC26 upstream of OPA1 in regulation of efferocytosis.","method":"Macrophage-specific KO mice (MIC26LysM); in vitro efferocytosis assay; in vivo thymus efferocytosis assay; OPA1 silencing and overexpression; mitochondrial morphology and membrane potential analysis","journal":"Atherosclerosis","confidence":"Medium","confidence_rationale":"Tier 2 — epistasis via rescue/overexpression of OPA1 with defined cellular phenotype; single lab","pmids":["37995600"],"is_preprint":false},{"year":2023,"finding":"Adipocyte-specific deletion of APOO disrupts mitochondrial inner membrane structure in brown adipocytes, impairs oxidative phosphorylation, shifts metabolism from oxidative to glycolytic, increases lipogenic enzyme levels, inhibits thermogenesis by reducing mitochondrial long-chain fatty acid oxidation, and disturbs peroxisomal biogenesis and very-long-chain fatty acid oxidation via PPARα.","method":"Adipocyte-specific KO mice (ApooACKO); mitochondrial ultrastructure by electron microscopy; OXPHOS measurement; metabolic flux analysis; cold exposure thermogenesis assay; peroxisome biogenesis assays; PPARα pathway analysis","journal":"Metabolism: clinical and experimental","confidence":"Medium","confidence_rationale":"Tier 2 — KO with defined mitochondrial and metabolic phenotypes; PPARα pathway identified; single lab","pmids":["37088120"],"is_preprint":false}],"current_model":"APOO encodes MIC26, a 22 kDa protein exclusively localized to the mitochondrial inner membrane as a bona fide subunit of the MICOS complex, where it physically interacts with MIC60, MIC27, and MIC10, promotes crista junction formation, antagonizes MIC10 oligomerization (opposite to MIC27), and cooperates with MIC27 to maintain cardiolipin levels, respiratory chain supercomplex integrity, and F1Fo-ATP synthase assembly; in non-mitochondrial contexts it promotes cholesterol efflux and its loss in macrophages increases efferocytosis via reduced OPA1 and increased mitochondrial fission."},"narrative":{"teleology":[{"year":2006,"claim":"The initial characterization identified APOO as a novel apolipoprotein promoting cholesterol efflux, localizing to lipid droplets, and bearing a chondroitin sulfate chain — establishing a secreted lipoprotein identity that would later be revised.","evidence":"Confocal immunofluorescence with perilipins, chondroitinase treatment, MTP inhibitor experiments, and cholesterol efflux assays in macrophages","pmids":["16956892"],"confidence":"Medium","gaps":["Lipid-droplet localization and secreted isoform identity were later shown to reflect nonspecific antibody signal","No mitochondrial function assessed","Cholesterol efflux mechanism not delineated"]},{"year":2015,"claim":"Discovery that MIC26 is a bona fide MICOS subunit that spans the inner mitochondrial membrane, physically interacts with MIC60/MIC27/MIC10, and is required for crista junction maintenance and mitochondrial respiration — fundamentally redefining the gene's primary function from secreted apolipoprotein to mitochondrial structural protein.","evidence":"Reciprocal co-immunoprecipitation with MICOS subunits; miRNA knockdown with rescue; electron microscopy of cristae; respiration assays in human cells","pmids":["25764979","26217776"],"confidence":"High","gaps":["Mechanism by which MIC26 and MIC27 antagonistically regulate each other's levels was not resolved","Role of cardiolipin in MIC26 function not yet addressed","No in vivo or organism-level data"]},{"year":2018,"claim":"MIC26 was shown to destabilize MIC10 oligomers, functioning antagonistically to MIC27 which stabilizes them — revealing opposing regulatory roles of the two lipid-binding MICOS subunits in controlling the oligomeric state of the membrane-bending MIC10 subunit.","evidence":"Blue-native PAGE analysis of MIC10 oligomeric state in yeast and mammalian cells with MIC26/MIC27 deletion","pmids":["29733859"],"confidence":"Medium","gaps":["Structural basis for opposing effects on MIC10 oligomerization unknown","Functional consequence of altered MIC10 oligomeric state on crista morphology not directly tested in this study","Single lab findings"]},{"year":2020,"claim":"Combined loss of MIC26 and MIC27 demonstrated their cooperative requirement for cardiolipin homeostasis, respiratory chain supercomplex stability, and F1Fo-ATP synthase assembly — with cardiolipin synthase overexpression rescuing supercomplex defects, establishing cardiolipin as the key mediator.","evidence":"Double-KO human cells; complexome profiling; STED nanoscopy; BN-PAGE; cardiolipin synthase overexpression rescue","pmids":["32788226"],"confidence":"High","gaps":["Direct mechanism by which MIC26/MIC27 maintain cardiolipin levels not identified","Whether MIC26 has lipid-binding or lipid-transfer activity is unresolved"]},{"year":2020,"claim":"Identification of a pathogenic I117T missense mutation in APOO established MIC26 as a disease gene, showing the mutation impairs protein import into the inner membrane and disrupts MICOS assembly and crista junctions, with conserved phenotypes in yeast and Drosophila orthologs.","evidence":"Patient fibroblast studies; whole-exome sequencing; protein import assays; electron microscopy; cross-species KO models","pmids":["32439808"],"confidence":"High","gaps":["Full clinical spectrum of APOO-related mitochondrial disease not delineated","Whether the import defect reflects a direct chaperone interaction or membrane insertion failure is unclear"]},{"year":2023,"claim":"Definitive resolution of the isoform controversy: MIC26 exists exclusively as a 22 kDa mitochondrial protein; the previously reported 55 kDa secreted glycoprotein isoform was an antibody artifact, established across four cell lines, four antibodies, and mass spectrometry of the misidentified band.","evidence":"CRISPR KO in four human cell lines; multiple antibodies; tagged overexpression; glycosylation site mutagenesis; mass spectrometry","pmids":["37279200"],"confidence":"High","gaps":["The earlier cholesterol efflux phenotype has not been re-evaluated in light of the isoform correction","Whether any non-mitochondrial MIC26 function persists under physiological conditions is unresolved"]},{"year":2023,"claim":"A second pathogenic mutation (E178*) confirmed the disease relevance and showed that even correctly localized but unstable MIC26 fails to rescue cristae architecture, demonstrating that steady-state protein levels are critical for function.","evidence":"Patient exome sequencing; protein stability assays; co-IP; electron microscopy in KO rescue cells","pmids":["37649161"],"confidence":"Medium","gaps":["Single-family study; broader genotype-phenotype correlation not yet available","Whether degradation is proteasome- or mitochondrial-protease-mediated is unknown"]},{"year":2023,"claim":"Tissue-specific KO studies revealed that MIC26 loss in adipocytes impairs thermogenesis and shifts metabolism to glycolysis via disrupted oxidative phosphorylation and PPARα-dependent peroxisomal biogenesis, while macrophage-specific loss reduces OPA1, increases fission, and enhances efferocytosis — extending MIC26 function to whole-organism metabolic physiology.","evidence":"Adipocyte-specific and macrophage-specific KO mice; electron microscopy; OXPHOS and metabolic flux analysis; cold exposure; OPA1 rescue experiments; in vivo efferocytosis assay","pmids":["37088120","37995600"],"confidence":"Medium","gaps":["Direct molecular link between MIC26 loss and OPA1 reduction not identified","Whether PPARα pathway involvement is direct or secondary to OXPHOS impairment is unclear","In vivo cardiovascular or disease relevance of efferocytosis phenotype not tested"]},{"year":null,"claim":"Key unresolved questions include the structural basis of MIC26 within the MICOS complex, whether MIC26 directly binds or transfers cardiolipin, and the full clinical spectrum of APOO-associated mitochondrial disease.","evidence":"","pmids":[],"confidence":"Low","gaps":["No high-resolution structure of MIC26 within MICOS exists","No direct lipid-binding assay for MIC26 has been reported","Genotype-phenotype correlation across APOO mutations remains limited to two families"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[1,4,5]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[1,4,5,6,7]}],"pathway":[{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[1,4,5]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[4,9]}],"complexes":["MICOS"],"partners":["MIC60","MIC27","MIC10","OPA1"],"other_free_text":[]},"mechanistic_narrative":"APOO encodes MIC26, a 22 kDa inner mitochondrial membrane protein that functions as a subunit of the MICOS (mitochondrial contact site and cristae organizing system) complex, where it is essential for crista junction formation, respiratory chain supercomplex integrity, and mitochondrial ultrastructure. MIC26 physically interacts with MIC60, MIC27, and MIC10, destabilizes MIC10 oligomers (opposite to MIC27), and cooperates with MIC27 to maintain cardiolipin levels, F1Fo-ATP synthase assembly, and oxidative phosphorylation capacity [PMID:25764979, PMID:29733859, PMID:32788226]. Tissue-specific deletion in brown adipocytes impairs thermogenesis and shifts metabolism from oxidative to glycolytic, while macrophage-specific loss reduces OPA1, increases mitochondrial fission, and enhances efferocytosis [PMID:37088120, PMID:37995600]. Pathogenic mutations in APOO (I117T, E178*) impair MIC26 import or stability, disrupt MICOS assembly and cristae architecture, and cause mitochondrial disease [PMID:32439808, PMID:37649161]."},"prefetch_data":{"uniprot":{"accession":"Q9BUR5","full_name":"MICOS complex subunit MIC26","aliases":["Apolipoprotein O","MICOS complex subunit MIC23","Protein FAM121B"],"length_aa":198,"mass_kda":22.3,"function":"Component of the MICOS complex, a large protein complex of the mitochondrial inner membrane that plays crucial roles in the maintenance of crista junctions, inner membrane architecture, and formation of contact sites to the outer membrane. Plays a crucial role in crista junction formation and mitochondrial function (PubMed:25764979). Can promote cardiac lipotoxicity by enhancing mitochondrial respiration and fatty acid metabolism in cardiac myoblasts (PubMed:24743151). Promotes cholesterol efflux from macrophage cells. Detected in HDL, LDL and VLDL. Secreted by a microsomal triglyceride transfer protein (MTTP)-dependent mechanism, probably as a VLDL-associated protein that is subsequently transferred to HDL (PubMed:16956892)","subcellular_location":"Mitochondrion inner membrane; Secreted; Mitochondrion; Golgi apparatus membrane; Endoplasmic reticulum membrane","url":"https://www.uniprot.org/uniprotkb/Q9BUR5/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/APOO","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"DNAJC11","stoichiometry":4.0}],"url":"https://opencell.sf.czbiohub.org/search/APOO","total_profiled":1310},"omim":[{"mim_id":"616658","title":"MITOCHONDRIAL CONTACT SITE AND CRISTAE ORGANIZING SYSTEM, 13-KD SUBUNIT; MICOS13","url":"https://www.omim.org/entry/616658"},{"mim_id":"616574","title":"MITOCHONDRIAL CONTACT SITE AND CRISTAE ORGANIZING SYSTEM, 10-KD SUBUNIT; MICOS10","url":"https://www.omim.org/entry/616574"},{"mim_id":"300955","title":"APOLIPOPROTEIN O-LIKE; APOOL","url":"https://www.omim.org/entry/300955"},{"mim_id":"300753","title":"APOLIPOPROTEIN O; APOO","url":"https://www.omim.org/entry/300753"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Mitochondria","reliability":"Supported"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"choroid plexus","ntpm":102.5}],"url":"https://www.proteinatlas.org/search/APOO"},"hgnc":{"alias_symbol":["MGC4825","My025","Mic23","MIC26","MICOS26"],"prev_symbol":["FAM121B"]},"alphafold":{"accession":"Q9BUR5","domains":[{"cath_id":"-","chopping":"57-129","consensus_level":"medium","plddt":86.5042,"start":57,"end":129},{"cath_id":"1.10.287","chopping":"132-183","consensus_level":"medium","plddt":76.8198,"start":132,"end":183}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BUR5","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BUR5-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BUR5-F1-predicted_aligned_error_v6.png","plddt_mean":74.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=APOO","jax_strain_url":"https://www.jax.org/strain/search?query=APOO"},"sequence":{"accession":"Q9BUR5","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9BUR5.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9BUR5/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BUR5"}},"corpus_meta":[{"pmid":"25764979","id":"PMC_25764979","title":"The non-glycosylated isoform of MIC26 is a constituent of the mammalian MICOS complex and promotes formation of crista junctions.","date":"2015","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/25764979","citation_count":72,"is_preprint":false},{"pmid":"16956892","id":"PMC_16956892","title":"ApoO, a novel apolipoprotein, is an original glycoprotein up-regulated by diabetes in human heart.","date":"2006","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/16956892","citation_count":61,"is_preprint":false},{"pmid":"29733859","id":"PMC_29733859","title":"Assembly of the Mitochondrial Cristae Organizer Mic10 Is Regulated by Mic26-Mic27 Antagonism and Cardiolipin.","date":"2018","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/29733859","citation_count":44,"is_preprint":false},{"pmid":"32788226","id":"PMC_32788226","title":"MIC26 and MIC27 cooperate to regulate cardiolipin levels and the landscape of OXPHOS complexes.","date":"2020","source":"Life science alliance","url":"https://pubmed.ncbi.nlm.nih.gov/32788226","citation_count":41,"is_preprint":false},{"pmid":"32439808","id":"PMC_32439808","title":"Mutation in the MICOS subunit gene APOO (MIC26) associated with an X-linked recessive mitochondrial myopathy, lactic acidosis, cognitive impairment and autistic features.","date":"2020","source":"Journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/32439808","citation_count":39,"is_preprint":false},{"pmid":"24391192","id":"PMC_24391192","title":"Novel intracellular functions of apolipoproteins: the ApoO protein family as constituents of the Mitofilin/MINOS complex determines cristae morphology in mitochondria.","date":"2014","source":"Biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/24391192","citation_count":29,"is_preprint":false},{"pmid":"37088120","id":"PMC_37088120","title":"Loss of APOO (MIC26) aggravates obesity-related whitening of brown adipose tissue via PPARα-mediated functional interplay between mitochondria and peroxisomes.","date":"2023","source":"Metabolism: clinical and experimental","url":"https://pubmed.ncbi.nlm.nih.gov/37088120","citation_count":13,"is_preprint":false},{"pmid":"37649161","id":"PMC_37649161","title":"A X-linked nonsense APOO/MIC26 variant causes a lethal mitochondrial disease with progeria-like phenotypes.","date":"2023","source":"Clinical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/37649161","citation_count":10,"is_preprint":false},{"pmid":"37995600","id":"PMC_37995600","title":"Macrophage-specific deletion of MIC26 (APOO) mitigates advanced atherosclerosis by increasing efferocytosis.","date":"2023","source":"Atherosclerosis","url":"https://pubmed.ncbi.nlm.nih.gov/37995600","citation_count":10,"is_preprint":false},{"pmid":"29593015","id":"PMC_29593015","title":"An APOO Pseudogene on Chromosome 5q Is Associated With Low-Density Lipoprotein Cholesterol Levels.","date":"2018","source":"Circulation","url":"https://pubmed.ncbi.nlm.nih.gov/29593015","citation_count":10,"is_preprint":false},{"pmid":"37279200","id":"PMC_37279200","title":"MIC26 and MIC27 are bona fide subunits of the MICOS complex in mitochondria and do not exist as glycosylated apolipoproteins.","date":"2023","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/37279200","citation_count":4,"is_preprint":false},{"pmid":"26217777","id":"PMC_26217777","title":"Data supporting the role of the non-glycosylated isoform of MIC26 in determining cristae morphology.","date":"2015","source":"Data in brief","url":"https://pubmed.ncbi.nlm.nih.gov/26217777","citation_count":3,"is_preprint":false},{"pmid":"21368419","id":"PMC_21368419","title":"Construction and expression of recombinant fusion protein of thioredoxin-ApoO.","date":"2011","source":"Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences","url":"https://pubmed.ncbi.nlm.nih.gov/21368419","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7805,"output_tokens":2703,"usd":0.03198},"stage2":{"model":"claude-opus-4-6","input_tokens":6034,"output_tokens":2489,"usd":0.138593},"total_usd":0.170573,"stage1_batch_id":"msgbatch_01TqNM6p6FsoMBT2jzQTwKBr","stage2_batch_id":"msgbatch_01Ya473eco8sTHbvRymQaU8o","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2006,\n      \"finding\": \"APOO (ApoO) is a novel apolipoprotein that colocalizes with perilipins at lipid droplets, belongs to the proteoglycan family (contains a chondroitin sulfate chain), requires microsomal triglyceride transfer protein (MTP) activity for secretion, and promotes cholesterol efflux from macrophages.\",\n      \"method\": \"Confocal immunofluorescence colocalization with perilipins; chondroitinase ABC deglycosylation; p-nitrophenyl-β-d-xyloside treatment; MTP inhibitor (CP-346086) treatment; cholesterol efflux assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal biochemical methods in a single lab establishing molecular properties and a functional activity\",\n      \"pmids\": [\"16956892\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The non-glycosylated 22 kDa isoform of MIC26 (APOO) spans the mitochondrial inner membrane and physically interacts with MICOS subunits MIC60, MIC27, and MIC10; its depletion reduces crista junction number and mitochondrial oxygen consumption, while overexpression induces mitochondrial fragmentation, ROS formation, and impaired respiration. MIC26 and MIC27 regulate each other's levels antagonistically, and both are positively correlated with MIC10 and tafazzin levels.\",\n      \"method\": \"Co-immunoprecipitation; miRNA-mediated knockdown; overexpression; electron microscopy of cristae ultrastructure; mitochondrial respiration assay; ROS measurement; confocal microscopy of mitochondrial network\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP with multiple MICOS subunits, loss-of-function with defined ultrastructural and functional phenotypes, replicated across multiple experiments\",\n      \"pmids\": [\"25764979\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"miRNA-mediated depletion of MIC26 increases MIC27 protein levels and decreases MIC10 levels; a miRNA-resistant form of MIC26 confirmed specificity. Depletion selectively reduces the 22 kDa mitochondrial isoform but not the 55 kDa secreted isoform; overexpression of myc-tagged MIC26 alters cristae morphology, producing swollen and vesicular cristae structures.\",\n      \"method\": \"miRNA knockdown with rescue; Western blot; electron microscopy\",\n      \"journal\": \"Data in brief\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — rescue experiment confirms specificity; single lab\",\n      \"pmids\": [\"26217776\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"MIC26 (APOO) exerts a destabilizing effect on MIC10 oligomers, functioning antagonistically to MIC27, which stabilizes MIC10 oligomers; cardiolipin also stabilizes MIC10 oligomers within the MICOS complex.\",\n      \"method\": \"Blue-native PAGE; biochemical analysis of Mic10 oligomeric state in yeast and mammalian cells with Mic26/Mic27 deletion\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic deletion with defined biochemical phenotype; single lab\",\n      \"pmids\": [\"29733859\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MIC26 and MIC27 are cooperatively required for integrity of respiratory chain supercomplexes and F1Fo-ATP synthase; double knockout (DKO) of MIC26 and MIC27 causes onion-like cristae with loss of crista junctions, reduces cardiolipin levels, and impairs integration of F1 subunits into monomeric F1Fo-ATP synthase. Overexpression of cardiolipin synthase in DKO rescues respiratory chain supercomplex stability. MIC26 and MIC27 assemble late into the MICOS complex and are dispensable for stability of remaining MICOS subunits.\",\n      \"method\": \"Single and double KO human cells; complexome profiling; STED nanoscopy; blue-native gel electrophoresis; cardiolipin synthase overexpression rescue\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal methods (complexome profiling, super-resolution microscopy, BN-PAGE), genetic rescue, single lab with rigorous controls\",\n      \"pmids\": [\"32788226\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"A missense mutation (I117T) in APOO/MIC26 impairs protein import and insertion into the inner mitochondrial membrane, causing altered MICOS assembly and crista junction disruption. Corresponding mutations in yeast and Drosophila MIC26 orthologs also cause mitochondrial structural and functional deficiencies, confirming a conserved role in MICOS assembly.\",\n      \"method\": \"Patient fibroblast studies; whole exome sequencing; protein import assays; electron microscopy; yeast and Drosophila knockout models\",\n      \"journal\": \"Journal of medical genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — pathogenic variant with mechanistic follow-up in human cells and two model organisms, multiple orthogonal methods\",\n      \"pmids\": [\"32439808\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"MIC26 (APOO) and MIC27 are exclusively localized to mitochondria as 22 kDa and 30 kDa proteins respectively; the previously reported 55 kDa glycosylated secreted MIC26 isoform is nonspecific, as confirmed by KO of MIC26 in four human cell lines using four different antibodies, GFP/myc-tagged overexpression, glycosylation site mutagenesis, and mass spectrometry.\",\n      \"method\": \"CRISPR KO in four human cell lines; multiple anti-MIC26 antibodies; tagged protein overexpression; glycosylation site mutagenesis; mass spectrometry of gel band\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal approaches in multiple cell lines definitively resolve isoform identity\",\n      \"pmids\": [\"37279200\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"A nonsense mutation (E178*) in APOO/MIC26 causing loss of 20 C-terminal amino acids produces a highly unstable, degradation-prone protein; the residual mutant MIC26 correctly localizes to mitochondria and interacts physically with other MICOS subunits, but MIC26 KO cells expressing this mutant show perturbed cristae architecture and fragmented mitochondria resembling full KO.\",\n      \"method\": \"Patient exome sequencing; protein stability assays; mitochondrial localization by immunofluorescence; co-immunoprecipitation; electron microscopy of cristae in KO rescue cells\",\n      \"journal\": \"Clinical genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple methods in patient-derived and engineered cells; single lab\",\n      \"pmids\": [\"37649161\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Macrophage-specific deletion of MIC26 (APOO) reduces mitochondrial OPA1 protein, increases mitochondrial fission, decreases mitochondrial membrane potential, and promotes efferocytosis; OPA1 overexpression abolishes the increased efferocytosis caused by MIC26 deficiency, placing MIC26 upstream of OPA1 in regulation of efferocytosis.\",\n      \"method\": \"Macrophage-specific KO mice (MIC26LysM); in vitro efferocytosis assay; in vivo thymus efferocytosis assay; OPA1 silencing and overexpression; mitochondrial morphology and membrane potential analysis\",\n      \"journal\": \"Atherosclerosis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — epistasis via rescue/overexpression of OPA1 with defined cellular phenotype; single lab\",\n      \"pmids\": [\"37995600\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Adipocyte-specific deletion of APOO disrupts mitochondrial inner membrane structure in brown adipocytes, impairs oxidative phosphorylation, shifts metabolism from oxidative to glycolytic, increases lipogenic enzyme levels, inhibits thermogenesis by reducing mitochondrial long-chain fatty acid oxidation, and disturbs peroxisomal biogenesis and very-long-chain fatty acid oxidation via PPARα.\",\n      \"method\": \"Adipocyte-specific KO mice (ApooACKO); mitochondrial ultrastructure by electron microscopy; OXPHOS measurement; metabolic flux analysis; cold exposure thermogenesis assay; peroxisome biogenesis assays; PPARα pathway analysis\",\n      \"journal\": \"Metabolism: clinical and experimental\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO with defined mitochondrial and metabolic phenotypes; PPARα pathway identified; single lab\",\n      \"pmids\": [\"37088120\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"APOO encodes MIC26, a 22 kDa protein exclusively localized to the mitochondrial inner membrane as a bona fide subunit of the MICOS complex, where it physically interacts with MIC60, MIC27, and MIC10, promotes crista junction formation, antagonizes MIC10 oligomerization (opposite to MIC27), and cooperates with MIC27 to maintain cardiolipin levels, respiratory chain supercomplex integrity, and F1Fo-ATP synthase assembly; in non-mitochondrial contexts it promotes cholesterol efflux and its loss in macrophages increases efferocytosis via reduced OPA1 and increased mitochondrial fission.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"APOO encodes MIC26, a 22 kDa inner mitochondrial membrane protein that functions as a subunit of the MICOS (mitochondrial contact site and cristae organizing system) complex, where it is essential for crista junction formation, respiratory chain supercomplex integrity, and mitochondrial ultrastructure. MIC26 physically interacts with MIC60, MIC27, and MIC10, destabilizes MIC10 oligomers (opposite to MIC27), and cooperates with MIC27 to maintain cardiolipin levels, F1Fo-ATP synthase assembly, and oxidative phosphorylation capacity [PMID:25764979, PMID:29733859, PMID:32788226]. Tissue-specific deletion in brown adipocytes impairs thermogenesis and shifts metabolism from oxidative to glycolytic, while macrophage-specific loss reduces OPA1, increases mitochondrial fission, and enhances efferocytosis [PMID:37088120, PMID:37995600]. Pathogenic mutations in APOO (I117T, E178*) impair MIC26 import or stability, disrupt MICOS assembly and cristae architecture, and cause mitochondrial disease [PMID:32439808, PMID:37649161].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"The initial characterization identified APOO as a novel apolipoprotein promoting cholesterol efflux, localizing to lipid droplets, and bearing a chondroitin sulfate chain — establishing a secreted lipoprotein identity that would later be revised.\",\n      \"evidence\": \"Confocal immunofluorescence with perilipins, chondroitinase treatment, MTP inhibitor experiments, and cholesterol efflux assays in macrophages\",\n      \"pmids\": [\"16956892\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Lipid-droplet localization and secreted isoform identity were later shown to reflect nonspecific antibody signal\",\n        \"No mitochondrial function assessed\",\n        \"Cholesterol efflux mechanism not delineated\"\n      ]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Discovery that MIC26 is a bona fide MICOS subunit that spans the inner mitochondrial membrane, physically interacts with MIC60/MIC27/MIC10, and is required for crista junction maintenance and mitochondrial respiration — fundamentally redefining the gene's primary function from secreted apolipoprotein to mitochondrial structural protein.\",\n      \"evidence\": \"Reciprocal co-immunoprecipitation with MICOS subunits; miRNA knockdown with rescue; electron microscopy of cristae; respiration assays in human cells\",\n      \"pmids\": [\"25764979\", \"26217776\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Mechanism by which MIC26 and MIC27 antagonistically regulate each other's levels was not resolved\",\n        \"Role of cardiolipin in MIC26 function not yet addressed\",\n        \"No in vivo or organism-level data\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"MIC26 was shown to destabilize MIC10 oligomers, functioning antagonistically to MIC27 which stabilizes them — revealing opposing regulatory roles of the two lipid-binding MICOS subunits in controlling the oligomeric state of the membrane-bending MIC10 subunit.\",\n      \"evidence\": \"Blue-native PAGE analysis of MIC10 oligomeric state in yeast and mammalian cells with MIC26/MIC27 deletion\",\n      \"pmids\": [\"29733859\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Structural basis for opposing effects on MIC10 oligomerization unknown\",\n        \"Functional consequence of altered MIC10 oligomeric state on crista morphology not directly tested in this study\",\n        \"Single lab findings\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Combined loss of MIC26 and MIC27 demonstrated their cooperative requirement for cardiolipin homeostasis, respiratory chain supercomplex stability, and F1Fo-ATP synthase assembly — with cardiolipin synthase overexpression rescuing supercomplex defects, establishing cardiolipin as the key mediator.\",\n      \"evidence\": \"Double-KO human cells; complexome profiling; STED nanoscopy; BN-PAGE; cardiolipin synthase overexpression rescue\",\n      \"pmids\": [\"32788226\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Direct mechanism by which MIC26/MIC27 maintain cardiolipin levels not identified\",\n        \"Whether MIC26 has lipid-binding or lipid-transfer activity is unresolved\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identification of a pathogenic I117T missense mutation in APOO established MIC26 as a disease gene, showing the mutation impairs protein import into the inner membrane and disrupts MICOS assembly and crista junctions, with conserved phenotypes in yeast and Drosophila orthologs.\",\n      \"evidence\": \"Patient fibroblast studies; whole-exome sequencing; protein import assays; electron microscopy; cross-species KO models\",\n      \"pmids\": [\"32439808\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Full clinical spectrum of APOO-related mitochondrial disease not delineated\",\n        \"Whether the import defect reflects a direct chaperone interaction or membrane insertion failure is unclear\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Definitive resolution of the isoform controversy: MIC26 exists exclusively as a 22 kDa mitochondrial protein; the previously reported 55 kDa secreted glycoprotein isoform was an antibody artifact, established across four cell lines, four antibodies, and mass spectrometry of the misidentified band.\",\n      \"evidence\": \"CRISPR KO in four human cell lines; multiple antibodies; tagged overexpression; glycosylation site mutagenesis; mass spectrometry\",\n      \"pmids\": [\"37279200\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The earlier cholesterol efflux phenotype has not been re-evaluated in light of the isoform correction\",\n        \"Whether any non-mitochondrial MIC26 function persists under physiological conditions is unresolved\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"A second pathogenic mutation (E178*) confirmed the disease relevance and showed that even correctly localized but unstable MIC26 fails to rescue cristae architecture, demonstrating that steady-state protein levels are critical for function.\",\n      \"evidence\": \"Patient exome sequencing; protein stability assays; co-IP; electron microscopy in KO rescue cells\",\n      \"pmids\": [\"37649161\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single-family study; broader genotype-phenotype correlation not yet available\",\n        \"Whether degradation is proteasome- or mitochondrial-protease-mediated is unknown\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Tissue-specific KO studies revealed that MIC26 loss in adipocytes impairs thermogenesis and shifts metabolism to glycolysis via disrupted oxidative phosphorylation and PPARα-dependent peroxisomal biogenesis, while macrophage-specific loss reduces OPA1, increases fission, and enhances efferocytosis — extending MIC26 function to whole-organism metabolic physiology.\",\n      \"evidence\": \"Adipocyte-specific and macrophage-specific KO mice; electron microscopy; OXPHOS and metabolic flux analysis; cold exposure; OPA1 rescue experiments; in vivo efferocytosis assay\",\n      \"pmids\": [\"37088120\", \"37995600\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct molecular link between MIC26 loss and OPA1 reduction not identified\",\n        \"Whether PPARα pathway involvement is direct or secondary to OXPHOS impairment is unclear\",\n        \"In vivo cardiovascular or disease relevance of efferocytosis phenotype not tested\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the structural basis of MIC26 within the MICOS complex, whether MIC26 directly binds or transfers cardiolipin, and the full clinical spectrum of APOO-associated mitochondrial disease.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No high-resolution structure of MIC26 within MICOS exists\",\n        \"No direct lipid-binding assay for MIC26 has been reported\",\n        \"Genotype-phenotype correlation across APOO mutations remains limited to two families\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [1, 4, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [1, 4, 5, 6, 7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:1852241\", \"supporting_discovery_ids\": [1, 4, 5]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [1, 4, 5]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [4, 9]}\n    ],\n    \"complexes\": [\n      \"MICOS\"\n    ],\n    \"partners\": [\n      \"MIC60\",\n      \"MIC27\",\n      \"MIC10\",\n      \"OPA1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}