{"gene":"APOO","run_date":"2026-06-09T22:02:44","timeline":{"discoveries":[{"year":2006,"finding":"ApoO (APOO) is a secreted glycoprotein that belongs to the proteoglycan family (contains chondroitin sulfate chains), co-localizes with perilipins at lipid droplets, promotes cholesterol efflux from macrophages, and requires microsomal triglyceride transfer protein (MTP) activity for secretion.","method":"Confocal immunofluorescence microscopy, chondroitinase ABC deglycosylation, xyloside treatment, naringenin/CP-346086 pharmacological inhibition of MTP, cholesterol efflux assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (co-localization, enzymatic deglycosylation, pharmacological inhibition, functional efflux assay) in a single lab","pmids":["16956892"],"is_preprint":false},{"year":2015,"finding":"The non-glycosylated 22 kDa mitochondrial isoform of MIC26 (APOO) spans the mitochondrial inner membrane, physically interacts with MICOS complex subunits MIC60, MIC27, and MIC10, and is required for crista junction formation; its depletion reduces crista junction number and alters mitochondrial ultrastructure.","method":"Co-immunoprecipitation, miRNA-mediated knockdown, electron microscopy, mitochondrial fractionation, immunofluorescence","journal":"Biochimica et biophysica acta","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP with multiple MICOS subunits, knockdown with defined ultrastructural phenotype, replicated by companion data paper (PMID:26217777)","pmids":["25764979","26217776"],"is_preprint":false},{"year":2015,"finding":"MIC26 and MIC27 (APOOL) regulate each other's protein levels in an antagonistic manner; overexpression of MIC26 increases MIC10 levels while depletion of MIC26 increases MIC27 levels. Both proteins are positively correlated with tafazzin levels, linking MIC26 to cardiolipin remodeling.","method":"miRNA-mediated knockdown, overexpression, immunoblotting with miRNA-resistant rescue constructs","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockdown with miRNA-resistant rescue (specificity control), single lab, multiple subunit measurements","pmids":["25764979","26217776"],"is_preprint":false},{"year":2015,"finding":"Overexpression of MIC26 induces mitochondrial fragmentation, promotes ROS formation, and impairs mitochondrial respiration; knockdown of MIC26 decreases mitochondrial oxygen consumption.","method":"Overexpression and miRNA-mediated knockdown, live-cell ROS measurement, oxygen consumption assay, mitochondrial network imaging","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss- and gain-of-function experiments with multiple functional readouts, single lab","pmids":["25764979"],"is_preprint":false},{"year":2018,"finding":"MIC26 destabilizes Mic10 oligomers in an antagonistic manner to MIC27 (which stabilizes Mic10 oligomers), demonstrating that MIC26 negatively regulates the oligomeric scaffold formed by the core MICOS subunit Mic10.","method":"Blue-native PAGE, biochemical fractionation, in vitro reconstitution/oligomerization assays, genetic manipulation in yeast","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — biochemical reconstitution of Mic10 oligomers with Mic26/Mic27, multiple orthogonal methods including cardiolipin supplementation, single lab","pmids":["29733859"],"is_preprint":false},{"year":2020,"finding":"MIC26 and MIC27 together are required for integrity of respiratory chain (super)complexes and F1Fo-ATP synthase (including integration of F1 subunits), and cooperatively regulate cardiolipin levels; restoring cardiolipin by overexpression of cardiolipin synthase in double knockout cells rescues respiratory chain supercomplex stability.","method":"Single and double knockout cell lines, complexome profiling, STED nanoscopy, blue-native gel electrophoresis, cardiolipin measurement, cardiolipin synthase overexpression rescue","journal":"Life science alliance","confidence":"High","confidence_rationale":"Tier 2 / Strong — double KO genetics, multiple orthogonal methods (complexome profiling, STED, BN-PAGE), rescue experiment with cardiolipin synthase overexpression","pmids":["32788226"],"is_preprint":false},{"year":2020,"finding":"MIC26 and MIC27 are dispensable for stability and integration of the remaining MICOS subunits into the complex, indicating they assemble late into MICOS.","method":"Double knockout human cells, complexome profiling, blue-native gel electrophoresis","journal":"Life science alliance","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — double KO with complexome profiling, single lab, multiple methods","pmids":["32788226"],"is_preprint":false},{"year":2020,"finding":"A missense mutation (I117T) in APOO impairs MIC26 import processing and insertion into the inner mitochondrial membrane, causing altered MICOS assembly and crista junction disruption; corresponding mutations in yeast and Drosophila models confirmed MIC26 involvement in MICOS assembly and mitochondrial function.","method":"Whole exome sequencing, patient-derived fibroblasts, protein import assay, MICOS co-immunoprecipitation, Drosophila and yeast knockout models, electron microscopy","journal":"Journal of medical genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — patient mutation with biochemical validation in fibroblasts, confirmed by two independent model organisms (yeast and Drosophila), multiple methods including import assay and crista ultrastructure","pmids":["32439808"],"is_preprint":false},{"year":2023,"finding":"A nonsense mutation (E178*) in APOO/MIC26 producing a truncated protein lacking 20 C-terminal amino acids results in a highly unstable protein; remaining mutant MIC26 correctly localizes to mitochondria and physically interacts with other MICOS subunits, but cannot restore normal cristae architecture in MIC26 KO cells.","method":"Exome sequencing, MIC26 KO cell complementation with mutant protein, immunoprecipitation of MICOS subunits, immunofluorescence localization, mitochondrial morphology analysis","journal":"Clinical genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO complementation with patient mutant, co-IP showing residual interactions, single lab with multiple methods","pmids":["37649161"],"is_preprint":false},{"year":2023,"finding":"MIC26 and MIC27 are exclusively mitochondrial proteins (22 kDa and 30 kDa respectively); the previously reported 55 kDa glycosylated secreted MIC26 isoform is a non-specific signal, not a genuine MIC26 protein form, as confirmed by knockout lines, four independent antibodies, epitope-tagged constructs, glycosylation site mutagenesis, and mass spectrometry.","method":"CRISPR/siRNA knockouts in four human cell lines, four anti-MIC26 antibodies, GFP/myc-tagged constructs, glycosylation site mutagenesis, mass spectrometry of excised gel bands","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal methods (KO in 4 cell lines, 4 antibodies, tagged constructs, mutagenesis, MS), single lab but extremely rigorous controls","pmids":["37279200"],"is_preprint":false},{"year":2023,"finding":"Adipocyte-specific APOO knockout mice show disrupted mitochondrial structure in brown adipocytes, impaired oxidative phosphorylation, shift from oxidative to glycolytic metabolism, increased lipogenic enzyme levels, reduced long-chain fatty acid oxidation, and disturbed peroxisomal biogenesis and very-long-chain fatty acid oxidation via PPARα.","method":"Adipocyte-specific APOO knockout mice (ApooACKO), electron microscopy, oxygen consumption assays, metabolic phenotyping, PPARα pathway analysis","journal":"Metabolism: clinical and experimental","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO mouse model with multiple functional readouts and pathway identification via PPARα, single lab","pmids":["37088120"],"is_preprint":false},{"year":2023,"finding":"Macrophage-specific MIC26 (APOO) deletion increases efferocytosis by reducing mitochondrial OPA1 protein levels (causing increased mitochondrial fission and reduced membrane potential); OPA1 silencing phenocopied the efferocytosis increase, and OPA1 overexpression abolished the efferocytosis enhancement caused by MIC26 deficiency.","method":"Macrophage-specific MIC26 knockout mice (MIC26LysM), in vitro efferocytosis assay, in vivo thymus efferocytosis assay, OPA1 siRNA knockdown and overexpression epistasis, mitochondrial membrane potential measurement, electron microscopy","journal":"Atherosclerosis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — macrophage-specific KO with OPA1 epistasis experiments (knockdown and rescue), multiple functional readouts, single lab","pmids":["37995600"],"is_preprint":false}],"current_model":"APOO (MIC26) encodes a 22 kDa inner mitochondrial membrane protein that is a bona fide subunit of the MICOS complex—interacting physically with MIC60, MIC27, and MIC10—where it promotes crista junction formation, destabilizes Mic10 oligomers antagonistically to MIC27, cooperates with MIC27 to maintain cardiolipin levels and the integrity of respiratory chain supercomplexes and ATP synthase, and influences mitochondrial dynamics and efferocytosis via regulation of OPA1; pathogenic mutations in APOO disrupt MICOS assembly and cause X-linked mitochondrial disease."},"narrative":{"mechanistic_narrative":"APOO (MIC26) encodes a 22 kDa integral inner mitochondrial membrane protein that functions as a subunit of the MICOS complex governing crista junction formation and mitochondrial ultrastructure [PMID:25764979, PMID:26217776]. It is incorporated into MICOS through physical interactions with MIC60, MIC27, and MIC10, and its depletion reduces crista junction number and distorts cristae architecture [PMID:25764979, PMID:26217776]. Within the complex MIC26 acts antagonistically to its paralog MIC27 (APOOL): it destabilizes the MIC10 oligomeric scaffold that MIC27 stabilizes, and the two proteins reciprocally regulate each other's levels [PMID:25764979, PMID:26217776, PMID:29733859]. MIC26 and MIC27 assemble late into MICOS and are dispensable for integration of the remaining subunits, but together they are required for cardiolipin homeostasis and for the integrity of respiratory chain supercomplexes and F1Fo-ATP synthase, with cardiolipin synthase overexpression rescuing supercomplex stability in double-knockout cells [PMID:32788226]. Through this role MIC26 shapes mitochondrial bioenergetics and physiology: adipocyte-specific loss impairs oxidative phosphorylation and shifts metabolism toward glycolysis [PMID:37088120], and macrophage-specific loss enhances efferocytosis by lowering OPA1 levels to drive mitochondrial fission [PMID:37995600]. Pathogenic APOO mutations—a missense I117T that impairs import and membrane insertion, and a C-terminal-truncating nonsense E178* that destabilizes the protein—disrupt MICOS assembly and cristae architecture, causing X-linked mitochondrial disease [PMID:32439808, PMID:37649161]. An earlier model of APOO as a secreted glycoprotein acting in cholesterol efflux [PMID:16956892] was shown to reflect non-specific signal rather than a genuine protein isoform; MIC26 is exclusively mitochondrial [PMID:37279200].","teleology":[{"year":2006,"claim":"The first functional assignment placed APOO outside the mitochondrion, proposing it as a secreted chondroitin-sulfate proteoglycan acting in macrophage cholesterol efflux.","evidence":"Confocal immunofluorescence, enzymatic deglycosylation, MTP pharmacological inhibition, and cholesterol efflux assays in a single lab","pmids":["16956892"],"confidence":"Medium","gaps":["The secreted glycoprotein assignment was later attributed to non-specific signal","No mitochondrial role identified at this stage"]},{"year":2015,"claim":"Identification of MIC26 as a MICOS subunit redefined APOO as an inner-membrane protein required for crista junction formation, answering where it acts and which complex it belongs to.","evidence":"Reciprocal co-immunoprecipitation with MIC60/MIC27/MIC10, miRNA knockdown, and electron microscopy of cristae, with companion data paper","pmids":["25764979","26217776"],"confidence":"High","gaps":["Molecular mechanism of crista junction formation not resolved","Stoichiometry within MICOS not defined"]},{"year":2015,"claim":"Antagonistic regulation of protein levels between MIC26 and MIC27 and correlation with tafazzin established a regulatory relationship linking MIC26 to cardiolipin remodeling.","evidence":"miRNA knockdown and overexpression with miRNA-resistant rescue constructs and immunoblotting of MICOS subunits","pmids":["25764979","26217776"],"confidence":"Medium","gaps":["Mechanism of reciprocal level regulation unknown","Direct effect on cardiolipin not yet demonstrated"]},{"year":2018,"claim":"Biochemical reconstitution defined the molecular action of MIC26 as destabilizing MIC10 oligomers, opposite to the stabilizing role of MIC27, clarifying how the two paralogs tune the MICOS scaffold.","evidence":"Blue-native PAGE, in vitro oligomerization assays, and genetic manipulation in yeast","pmids":["29733859"],"confidence":"High","gaps":["Structural basis of MIC10 oligomer destabilization unresolved","Quantitative balance of MIC26/MIC27 in vivo not established"]},{"year":2020,"claim":"Double-knockout genetics linked MIC26/MIC27 to cardiolipin homeostasis and respiratory chain supercomplex and ATP synthase integrity, and showed they assemble late into MICOS, connecting MICOS architecture to bioenergetic assembly.","evidence":"Single/double knockout cells, complexome profiling, STED nanoscopy, BN-PAGE, and cardiolipin synthase overexpression rescue","pmids":["32788226"],"confidence":"High","gaps":["Direct mechanism by which cardiolipin loss destabilizes supercomplexes not detailed","Individual contribution of MIC26 versus MIC27 partly redundant"]},{"year":2020,"claim":"A patient missense mutation establishing APOO as a cause of X-linked mitochondrial disease, by showing impaired import and MICOS assembly defects validated across model organisms.","evidence":"Whole exome sequencing, patient fibroblast import assays, MICOS co-IP, and yeast and Drosophila models with EM","pmids":["32439808"],"confidence":"High","gaps":["Genotype-phenotype spectrum across patients not defined","How import failure translates to organ-level disease unclear"]},{"year":2023,"claim":"A second pathogenic allele (E178* truncation) showed that loss of the C-terminus destabilizes MIC26 while preserving its MICOS interactions, distinguishing protein stability from complex binding in disease.","evidence":"Exome sequencing, KO complementation with mutant protein, co-IP of MICOS subunits, and morphology analysis","pmids":["37649161"],"confidence":"Medium","gaps":["Why residual interacting mutant fails to restore cristae unexplained","Single patient context"]},{"year":2023,"claim":"Rigorous knockout and antibody controls overturned the original secreted-glycoprotein model, establishing MIC26 as an exclusively mitochondrial protein.","evidence":"CRISPR/siRNA knockouts in four cell lines, four antibodies, tagged constructs, glycosylation-site mutagenesis, and mass spectrometry of gel bands","pmids":["37279200"],"confidence":"High","gaps":["Identity of the misattributed 55 kDa band not fully resolved"]},{"year":2023,"claim":"Tissue-specific knockout mice extended MIC26 function to physiology, showing roles in adipocyte oxidative metabolism and in macrophage efferocytosis via OPA1.","evidence":"Adipocyte- and macrophage-specific knockout mice with metabolic phenotyping, EM, PPARalpha analysis, and OPA1 epistasis (knockdown and overexpression)","pmids":["37088120","37995600"],"confidence":"Medium","gaps":["Mechanism connecting MIC26 loss to OPA1 reduction unknown","Generalizability of metabolic shift across tissues untested"]},{"year":null,"claim":"How MIC26 mechanistically links MICOS architecture and cardiolipin to downstream regulators such as OPA1 and to tissue-specific metabolic outcomes remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of MIC26 within MICOS","Mechanism of OPA1 regulation by MIC26 undefined","Full clinical spectrum of APOO mutations not characterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[1,4]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[1,9]}],"pathway":[{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[1,5]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[5,10]}],"complexes":["MICOS complex"],"partners":["MIC60","MIC27","MIC10"],"other_free_text":[]}},"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":62,"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":46,"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":42,"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":40,"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":30,"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":16,"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":12,"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":11,"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":8582,"output_tokens":2993,"usd":0.03532,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10475,"output_tokens":3197,"usd":0.06615,"stage2_stop_reason":"end_turn"},"total_usd":0.10147,"stage1_batch_id":"msgbatch_0179Z2pxRYBsXi2xADneafRw","stage2_batch_id":"msgbatch_01LaY9ZaVdgjWArcZo9eqQE5","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2006,\n      \"finding\": \"ApoO (APOO) is a secreted glycoprotein that belongs to the proteoglycan family (contains chondroitin sulfate chains), co-localizes with perilipins at lipid droplets, promotes cholesterol efflux from macrophages, and requires microsomal triglyceride transfer protein (MTP) activity for secretion.\",\n      \"method\": \"Confocal immunofluorescence microscopy, chondroitinase ABC deglycosylation, xyloside treatment, naringenin/CP-346086 pharmacological inhibition of MTP, cholesterol efflux assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (co-localization, enzymatic deglycosylation, pharmacological inhibition, functional efflux assay) in a single lab\",\n      \"pmids\": [\"16956892\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The non-glycosylated 22 kDa mitochondrial isoform of MIC26 (APOO) spans the mitochondrial inner membrane, physically interacts with MICOS complex subunits MIC60, MIC27, and MIC10, and is required for crista junction formation; its depletion reduces crista junction number and alters mitochondrial ultrastructure.\",\n      \"method\": \"Co-immunoprecipitation, miRNA-mediated knockdown, electron microscopy, mitochondrial fractionation, immunofluorescence\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP with multiple MICOS subunits, knockdown with defined ultrastructural phenotype, replicated by companion data paper (PMID:26217777)\",\n      \"pmids\": [\"25764979\", \"26217776\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"MIC26 and MIC27 (APOOL) regulate each other's protein levels in an antagonistic manner; overexpression of MIC26 increases MIC10 levels while depletion of MIC26 increases MIC27 levels. Both proteins are positively correlated with tafazzin levels, linking MIC26 to cardiolipin remodeling.\",\n      \"method\": \"miRNA-mediated knockdown, overexpression, immunoblotting with miRNA-resistant rescue constructs\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockdown with miRNA-resistant rescue (specificity control), single lab, multiple subunit measurements\",\n      \"pmids\": [\"25764979\", \"26217776\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Overexpression of MIC26 induces mitochondrial fragmentation, promotes ROS formation, and impairs mitochondrial respiration; knockdown of MIC26 decreases mitochondrial oxygen consumption.\",\n      \"method\": \"Overexpression and miRNA-mediated knockdown, live-cell ROS measurement, oxygen consumption assay, mitochondrial network imaging\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss- and gain-of-function experiments with multiple functional readouts, single lab\",\n      \"pmids\": [\"25764979\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"MIC26 destabilizes Mic10 oligomers in an antagonistic manner to MIC27 (which stabilizes Mic10 oligomers), demonstrating that MIC26 negatively regulates the oligomeric scaffold formed by the core MICOS subunit Mic10.\",\n      \"method\": \"Blue-native PAGE, biochemical fractionation, in vitro reconstitution/oligomerization assays, genetic manipulation in yeast\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — biochemical reconstitution of Mic10 oligomers with Mic26/Mic27, multiple orthogonal methods including cardiolipin supplementation, single lab\",\n      \"pmids\": [\"29733859\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MIC26 and MIC27 together are required for integrity of respiratory chain (super)complexes and F1Fo-ATP synthase (including integration of F1 subunits), and cooperatively regulate cardiolipin levels; restoring cardiolipin by overexpression of cardiolipin synthase in double knockout cells rescues respiratory chain supercomplex stability.\",\n      \"method\": \"Single and double knockout cell lines, complexome profiling, STED nanoscopy, blue-native gel electrophoresis, cardiolipin measurement, cardiolipin synthase overexpression rescue\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — double KO genetics, multiple orthogonal methods (complexome profiling, STED, BN-PAGE), rescue experiment with cardiolipin synthase overexpression\",\n      \"pmids\": [\"32788226\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MIC26 and MIC27 are dispensable for stability and integration of the remaining MICOS subunits into the complex, indicating they assemble late into MICOS.\",\n      \"method\": \"Double knockout human cells, complexome profiling, blue-native gel electrophoresis\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — double KO with complexome profiling, single lab, multiple methods\",\n      \"pmids\": [\"32788226\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"A missense mutation (I117T) in APOO impairs MIC26 import processing and insertion into the inner mitochondrial membrane, causing altered MICOS assembly and crista junction disruption; corresponding mutations in yeast and Drosophila models confirmed MIC26 involvement in MICOS assembly and mitochondrial function.\",\n      \"method\": \"Whole exome sequencing, patient-derived fibroblasts, protein import assay, MICOS co-immunoprecipitation, Drosophila and yeast knockout models, electron microscopy\",\n      \"journal\": \"Journal of medical genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — patient mutation with biochemical validation in fibroblasts, confirmed by two independent model organisms (yeast and Drosophila), multiple methods including import assay and crista ultrastructure\",\n      \"pmids\": [\"32439808\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"A nonsense mutation (E178*) in APOO/MIC26 producing a truncated protein lacking 20 C-terminal amino acids results in a highly unstable protein; remaining mutant MIC26 correctly localizes to mitochondria and physically interacts with other MICOS subunits, but cannot restore normal cristae architecture in MIC26 KO cells.\",\n      \"method\": \"Exome sequencing, MIC26 KO cell complementation with mutant protein, immunoprecipitation of MICOS subunits, immunofluorescence localization, mitochondrial morphology analysis\",\n      \"journal\": \"Clinical genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO complementation with patient mutant, co-IP showing residual interactions, single lab with multiple methods\",\n      \"pmids\": [\"37649161\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"MIC26 and MIC27 are exclusively mitochondrial proteins (22 kDa and 30 kDa respectively); the previously reported 55 kDa glycosylated secreted MIC26 isoform is a non-specific signal, not a genuine MIC26 protein form, as confirmed by knockout lines, four independent antibodies, epitope-tagged constructs, glycosylation site mutagenesis, and mass spectrometry.\",\n      \"method\": \"CRISPR/siRNA knockouts in four human cell lines, four anti-MIC26 antibodies, GFP/myc-tagged constructs, glycosylation site mutagenesis, mass spectrometry of excised gel bands\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal methods (KO in 4 cell lines, 4 antibodies, tagged constructs, mutagenesis, MS), single lab but extremely rigorous controls\",\n      \"pmids\": [\"37279200\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Adipocyte-specific APOO knockout mice show disrupted mitochondrial structure in brown adipocytes, impaired oxidative phosphorylation, shift from oxidative to glycolytic metabolism, increased lipogenic enzyme levels, reduced long-chain fatty acid oxidation, and disturbed peroxisomal biogenesis and very-long-chain fatty acid oxidation via PPARα.\",\n      \"method\": \"Adipocyte-specific APOO knockout mice (ApooACKO), electron microscopy, oxygen consumption assays, metabolic phenotyping, PPARα pathway analysis\",\n      \"journal\": \"Metabolism: clinical and experimental\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO mouse model with multiple functional readouts and pathway identification via PPARα, single lab\",\n      \"pmids\": [\"37088120\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Macrophage-specific MIC26 (APOO) deletion increases efferocytosis by reducing mitochondrial OPA1 protein levels (causing increased mitochondrial fission and reduced membrane potential); OPA1 silencing phenocopied the efferocytosis increase, and OPA1 overexpression abolished the efferocytosis enhancement caused by MIC26 deficiency.\",\n      \"method\": \"Macrophage-specific MIC26 knockout mice (MIC26LysM), in vitro efferocytosis assay, in vivo thymus efferocytosis assay, OPA1 siRNA knockdown and overexpression epistasis, mitochondrial membrane potential measurement, electron microscopy\",\n      \"journal\": \"Atherosclerosis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — macrophage-specific KO with OPA1 epistasis experiments (knockdown and rescue), multiple functional readouts, single lab\",\n      \"pmids\": [\"37995600\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"APOO (MIC26) encodes a 22 kDa inner mitochondrial membrane protein that is a bona fide subunit of the MICOS complex—interacting physically with MIC60, MIC27, and MIC10—where it promotes crista junction formation, destabilizes Mic10 oligomers antagonistically to MIC27, cooperates with MIC27 to maintain cardiolipin levels and the integrity of respiratory chain supercomplexes and ATP synthase, and influences mitochondrial dynamics and efferocytosis via regulation of OPA1; pathogenic mutations in APOO disrupt MICOS assembly and cause X-linked mitochondrial disease.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"APOO (MIC26) encodes a 22 kDa integral inner mitochondrial membrane protein that functions as a subunit of the MICOS complex governing crista junction formation and mitochondrial ultrastructure [#1]. It is incorporated into MICOS through physical interactions with MIC60, MIC27, and MIC10, and its depletion reduces crista junction number and distorts cristae architecture [#1]. Within the complex MIC26 acts antagonistically to its paralog MIC27 (APOOL): it destabilizes the MIC10 oligomeric scaffold that MIC27 stabilizes, and the two proteins reciprocally regulate each other's levels [#2, #4]. MIC26 and MIC27 assemble late into MICOS and are dispensable for integration of the remaining subunits, but together they are required for cardiolipin homeostasis and for the integrity of respiratory chain supercomplexes and F1Fo-ATP synthase, with cardiolipin synthase overexpression rescuing supercomplex stability in double-knockout cells [#5, #6]. Through this role MIC26 shapes mitochondrial bioenergetics and physiology: adipocyte-specific loss impairs oxidative phosphorylation and shifts metabolism toward glycolysis [#10], and macrophage-specific loss enhances efferocytosis by lowering OPA1 levels to drive mitochondrial fission [#11]. Pathogenic APOO mutations—a missense I117T that impairs import and membrane insertion, and a C-terminal-truncating nonsense E178* that destabilizes the protein—disrupt MICOS assembly and cristae architecture, causing X-linked mitochondrial disease [#7, #8]. An earlier model of APOO as a secreted glycoprotein acting in cholesterol efflux [#0] was shown to reflect non-specific signal rather than a genuine protein isoform; MIC26 is exclusively mitochondrial [#9].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"The first functional assignment placed APOO outside the mitochondrion, proposing it as a secreted chondroitin-sulfate proteoglycan acting in macrophage cholesterol efflux.\",\n      \"evidence\": \"Confocal immunofluorescence, enzymatic deglycosylation, MTP pharmacological inhibition, and cholesterol efflux assays in a single lab\",\n      \"pmids\": [\"16956892\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The secreted glycoprotein assignment was later attributed to non-specific signal\", \"No mitochondrial role identified at this stage\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identification of MIC26 as a MICOS subunit redefined APOO as an inner-membrane protein required for crista junction formation, answering where it acts and which complex it belongs to.\",\n      \"evidence\": \"Reciprocal co-immunoprecipitation with MIC60/MIC27/MIC10, miRNA knockdown, and electron microscopy of cristae, with companion data paper\",\n      \"pmids\": [\"25764979\", \"26217776\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism of crista junction formation not resolved\", \"Stoichiometry within MICOS not defined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Antagonistic regulation of protein levels between MIC26 and MIC27 and correlation with tafazzin established a regulatory relationship linking MIC26 to cardiolipin remodeling.\",\n      \"evidence\": \"miRNA knockdown and overexpression with miRNA-resistant rescue constructs and immunoblotting of MICOS subunits\",\n      \"pmids\": [\"25764979\", \"26217776\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of reciprocal level regulation unknown\", \"Direct effect on cardiolipin not yet demonstrated\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Biochemical reconstitution defined the molecular action of MIC26 as destabilizing MIC10 oligomers, opposite to the stabilizing role of MIC27, clarifying how the two paralogs tune the MICOS scaffold.\",\n      \"evidence\": \"Blue-native PAGE, in vitro oligomerization assays, and genetic manipulation in yeast\",\n      \"pmids\": [\"29733859\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of MIC10 oligomer destabilization unresolved\", \"Quantitative balance of MIC26/MIC27 in vivo not established\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Double-knockout genetics linked MIC26/MIC27 to cardiolipin homeostasis and respiratory chain supercomplex and ATP synthase integrity, and showed they assemble late into MICOS, connecting MICOS architecture to bioenergetic assembly.\",\n      \"evidence\": \"Single/double knockout cells, complexome profiling, STED nanoscopy, BN-PAGE, and cardiolipin synthase overexpression rescue\",\n      \"pmids\": [\"32788226\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct mechanism by which cardiolipin loss destabilizes supercomplexes not detailed\", \"Individual contribution of MIC26 versus MIC27 partly redundant\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"A patient missense mutation establishing APOO as a cause of X-linked mitochondrial disease, by showing impaired import and MICOS assembly defects validated across model organisms.\",\n      \"evidence\": \"Whole exome sequencing, patient fibroblast import assays, MICOS co-IP, and yeast and Drosophila models with EM\",\n      \"pmids\": [\"32439808\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Genotype-phenotype spectrum across patients not defined\", \"How import failure translates to organ-level disease unclear\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"A second pathogenic allele (E178* truncation) showed that loss of the C-terminus destabilizes MIC26 while preserving its MICOS interactions, distinguishing protein stability from complex binding in disease.\",\n      \"evidence\": \"Exome sequencing, KO complementation with mutant protein, co-IP of MICOS subunits, and morphology analysis\",\n      \"pmids\": [\"37649161\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Why residual interacting mutant fails to restore cristae unexplained\", \"Single patient context\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Rigorous knockout and antibody controls overturned the original secreted-glycoprotein model, establishing MIC26 as an exclusively mitochondrial protein.\",\n      \"evidence\": \"CRISPR/siRNA knockouts in four cell lines, four antibodies, tagged constructs, glycosylation-site mutagenesis, and mass spectrometry of gel bands\",\n      \"pmids\": [\"37279200\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the misattributed 55 kDa band not fully resolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Tissue-specific knockout mice extended MIC26 function to physiology, showing roles in adipocyte oxidative metabolism and in macrophage efferocytosis via OPA1.\",\n      \"evidence\": \"Adipocyte- and macrophage-specific knockout mice with metabolic phenotyping, EM, PPARalpha analysis, and OPA1 epistasis (knockdown and overexpression)\",\n      \"pmids\": [\"37088120\", \"37995600\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism connecting MIC26 loss to OPA1 reduction unknown\", \"Generalizability of metabolic shift across tissues untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How MIC26 mechanistically links MICOS architecture and cardiolipin to downstream regulators such as OPA1 and to tissue-specific metabolic outcomes remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of MIC26 within MICOS\", \"Mechanism of OPA1 regulation by MIC26 undefined\", \"Full clinical spectrum of APOO mutations not characterized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [1, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005743\", \"supporting_discovery_ids\": [1, 9]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [1, 9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [1, 5]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [5, 10]}\n    ],\n    \"complexes\": [\"MICOS complex\"],\n    \"partners\": [\"MIC60\", \"MIC27\", \"MIC10\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":{"gene":"APOO","tier":"GROUNDING","verdict":"Evidence-grounding concern","subtype":"fabrication","uniprot_band":"medium","rules_fired":"R7","issue":"R7: fabricated (no corpus paper): 26217776"},"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}