{"gene":"AFG3L2","run_date":"2026-06-09T22:02:42","timeline":{"discoveries":[{"year":1999,"finding":"AFG3L2 encodes a 797-amino-acid mitochondrial protein highly homologous to yeast Afg3p and Rca1p; immunofluorescence studies demonstrated that AFG3L2 and paraplegin share the same subcellular localization in the mitochondrial compartment, and AFG3L2 was mapped to chromosome 18p11.","method":"Immunofluorescence, EST database screening, radiation hybrid mapping","journal":"Genomics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization by immunofluorescence, single lab, two orthogonal methods (immunofluorescence + mapping)","pmids":["10395799"],"is_preprint":false},{"year":2008,"finding":"AFG3L2 assembles with paraplegin into a supracomplex in the inner mitochondrial membrane responsible for mitochondrial protein quality control; unlike paraplegin (which only hetero-oligomerizes), AFG3L2 supports both homo-oligomerization and hetero-oligomerization, explaining its greater neuronal importance. Loss of AFG3L2 in mice causes marked impairment of axonal development with delayed myelination and poor axonal radial growth, leading to lethality at P16.","method":"Null and spontaneous missense mutant mouse models, histological analysis, immunofluorescence","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — two independent murine models (null and missense) with defined cellular phenotypes and molecular mechanism (homo- vs hetero-oligomerization) replicated across models","pmids":["18337413"],"is_preprint":false},{"year":2009,"finding":"Haploinsufficiency of Afg3l2 in mice causes respiratory chain dysfunction, increased reactive oxygen species production, and dark degeneration of Purkinje cells, establishing a mitochondria-mediated pathogenic mechanism for SCA28.","method":"Afg3l2 haploinsufficient mouse model, electron microscopy, ROS measurement, histology","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetically defined haploinsufficient mouse model with multiple orthogonal mechanistic readouts (ROS, respiratory chain, cell degeneration)","pmids":["19625515"],"is_preprint":false},{"year":2010,"finding":"Heterozygous missense mutations in AFG3L2 cause SCA28; m-AAA-deficient yeast expressing mutated human AFG3L2 homocomplex show respiratory deficiency, proteolytic impairment, and deficiency of respiratory chain complex IV. Mutations cluster in the proteolytic domain and are predicted by homology modeling to affect substrate handling.","method":"Yeast complementation assay, respiratory growth assay, structure homology modeling, Sanger sequencing in families","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — yeast functional reconstitution with proteolytic and respiratory readouts, replicated in multiple independent families, supported by structural modeling","pmids":["20208537"],"is_preprint":false},{"year":2011,"finding":"A homozygous AFG3L2 Y616C mutation causes recessive spastic ataxia-neuropathy; the Y616C variant is hypomorphic, exhibiting oligomerization defects both in yeast complementation assays and in patient fibroblasts — specifically, formation of AFG3L2(Y616C) homo-complexes and hetero-complexes with paraplegin is impaired.","method":"Yeast complementation assay, Blue Native PAGE in patient fibroblasts, whole-exome sequencing","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — yeast complementation plus patient fibroblast biochemistry, two orthogonal methods demonstrating oligomerization defect","pmids":["22022284"],"is_preprint":false},{"year":2012,"finding":"Conditional knockout of Afg3l2 in mouse Purkinje cells causes cell-autonomous neurodegeneration with early mitochondrial fragmentation and altered distribution in the dendritic tree. Constitutive knockout reveals a decreased rate of mitochondrial protein synthesis associated with impaired mitochondrial ribosome assembly, establishing defective mitochondrial protein synthesis as a central causative mechanism.","method":"Conditional and constitutive Afg3l2 knockout mouse models, live imaging, electron microscopy, mitochondrial protein synthesis assay, ribosome assembly analysis","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — cell-autonomous KO with multiple orthogonal mechanistic readouts (protein synthesis rate, ribosome assembly, mitochondrial morphology) across two mouse models","pmids":["23041622"],"is_preprint":false},{"year":2012,"finding":"Loss of AFG3L2 in mouse embryonic fibroblasts reduces mitochondrial Ca2+ uptake capacity, caused by fragmentation of the mitochondrial network secondary to respiratory dysfunction and consequent OPA1 processing. Overexpression of OPA1 in Afg3l2-/- MEFs rescues impaired mitochondrial Ca2+ buffering but fails to restore respiration.","method":"Afg3l2 KO MEFs, mitochondrial Ca2+ imaging, permeabilized cell assays, OPA1 overexpression rescue experiment","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Ca2+ imaging, rescue overexpression, permeabilized cells) establishing the OPA1-fragmentation-Ca2+ axis","pmids":["22678058"],"is_preprint":false},{"year":2014,"finding":"AFG3L2 regulates SPG7 (paraplegin) processing: SPG7 is cleaved and activated by AFG3L2 upon assembly into the mAAA protease complex. This processing is regulated by tyrosine phosphorylation of AFG3L2; the SPG7 Q688 variant bypasses this regulation and constitutively activates SPG7 mAAA protease, resulting in elevated ATP production and ROS.","method":"Biochemical processing assays in cell lines, phosphorylation studies, mitochondrial ROS and ATP measurements","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Moderate — direct biochemical demonstration of AFG3L2-mediated SPG7 cleavage and phosphorylation-dependent regulation with multiple functional readouts","pmids":["24767997"],"is_preprint":false},{"year":2014,"finding":"In SCA28 mice, mitochondria in Afg3l2-deficient Purkinje cells fail to buffer evoked Ca2+ peaks, resulting in enhanced cytoplasmic Ca2+ concentrations triggering dark degeneration. This Ca2+ handling defect results from negative synergism between mitochondrial depolarization and altered organelle trafficking to dendrites. Partial genetic silencing of mGluR1 or ceftriaxone-mediated glutamate clearance rescued the ataxic phenotype.","method":"Afg3l2 haploinsufficient mouse model, Ca2+ imaging in cultured PCs, genetic mGluR1 silencing, pharmacological rescue with ceftriaxone","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct Ca2+ imaging with mechanistic dissection plus two independent rescue strategies (genetic and pharmacological) in vivo and in vitro","pmids":["25485680"],"is_preprint":false},{"year":2018,"finding":"AFG3L2 contains multiple substrate specificity mechanisms: conserved residues in the presequence of mitochondrial ribosomal protein MrpL32 serve as a degron targeting it for processing by AFG3L2. Peptidase specificity profiling using mass spectrometry reveals constrained product lengths with strong preference for hydrophobic and small polar residues at the P1' position.","method":"Solubilized AFG3L2 in vitro assay, mass spectrometry-based peptidase specificity profiling, fluorogenic reporter peptides, mutagenesis of degron sequences","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic reconstitution with mutagenesis, MS-based specificity profiling, and functional validation with multiple substrates, single lab","pmids":["29932645"],"is_preprint":false},{"year":2018,"finding":"Loss of AFG3L2 and YME1L, individually and in combination, results in diminished cell proliferation, mitochondrial reticulum fragmentation, altered cristae morphogenesis, and defective respiratory chain biogenesis. AFG3L2 knockdown specifically impairs assembly and function of complex IV, while YME1L loss impairs complex I. Double knockdown causes marked OPA1 short form accumulation.","method":"siRNA knockdown, Blue Native PAGE, electron microscopy, respiratory chain activity assays, Western blot","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple knockdown conditions with orthogonal readouts, single lab","pmids":["30544562"],"is_preprint":false},{"year":2018,"finding":"In a patient carrying AFG3L2 p.R468C with concurrent SPG7 deletion, patient fibroblasts show abnormal OPA1 processing with mitochondrial network fragmentation — a phenotype not seen in SCA28 or SPG7 patients' cells alone. Yeast functional analysis confirmed pathogenicity of AFG3L2 p.R468C and revealed its distinct pathogenic mechanism from classic SCA28 mutations.","method":"Yeast complementation assay, patient fibroblast OPA1 processing analysis, mitochondrial morphology assessment","journal":"Human mutation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast complementation plus patient cell biochemistry, single lab, two methods","pmids":["30252181"],"is_preprint":false},{"year":2019,"finding":"Cryo-EM structure of the substrate-bound catalytic core of human AFG3L2 reveals multiple specialized structural features that integrate with conserved AAA+ motifs for ATP-dependent substrate translocation, unfolding, and degradation. Disease-relevant mutations localize to these unique structural features and distinctly influence AFG3L2 activity and stability.","method":"Cryo-electron microscopy, mutagenesis of disease-associated residues, enzymatic activity assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure with functional validation by mutagenesis and activity assays, providing molecular basis for disease mutations","pmids":["31327635"],"is_preprint":false},{"year":2019,"finding":"SCA28 patient fibroblasts carrying proteolytic domain missense mutations show inefficient mitochondrial fusion caused by increased OPA1 processing operated by hyperactivated OMA1. Altered mitochondrial proteostasis triggers OMA1 activation, and pharmacological attenuation of mitochondrial protein synthesis stabilizes OMA1 and OPA1 long forms, rescuing mitochondrial fusion efficiency.","method":"Patient fibroblasts, CRISPR/Cas9 AFG3L2 KO HEK293T cells, Afg3l2-/- MEFs, OPA1/OMA1 Western blot, Blue Native PAGE, mitochondrial Ca2+ measurement, chloramphenicol treatment rescue","journal":"Journal of medical genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple patient cell lines plus isogenic KO models, pharmacological rescue, and mechanistic dissection of OMA1-OPA1 axis, single lab but multiple orthogonal methods","pmids":["30910913"],"is_preprint":false},{"year":2020,"finding":"AFG3L2 mutations in the ATPase domain (distinct from the proteolytic domain mutations causing SCA28) cause dominant optic atrophy through a different mechanism: patient fibroblasts show abnormal OPA1 processing with accumulation of fission-inducing short forms and mitochondrial network fragmentation. Pathogenicity confirmed in yeast.","method":"Targeted NGS/WES, yeast complementation assay, patient fibroblast OPA1 processing analysis, mitochondrial morphology assessment","journal":"Annals of neurology","confidence":"High","confidence_rationale":"Tier 2 / Strong — yeast functional validation plus patient fibroblast biochemistry, mechanistic distinction from SCA28 established with multiple methods","pmids":["32219868"],"is_preprint":false},{"year":2020,"finding":"A novel AFG3L2 mutation (p.G337E) close to the AAA domain strongly destabilizes OPA1 long isoforms via OMA1 hyperactivation, leading to mitochondrial fragmentation in patient fibroblasts — a mechanism similar to ATPase-domain DOA mutations but distinct from proteolytic-domain SCA28 mutations.","method":"Patient fibroblast functional studies, OPA1/OMA1 Western blot, mitochondrial morphology assessment","journal":"Acta neuropathologica communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct patient fibroblast experiments with OMA1/OPA1 biochemical readouts, single lab, two orthogonal methods","pmids":["32600459"],"is_preprint":false},{"year":2018,"finding":"A knock-in mouse model carrying the SCA28 patient-derived Afg3l2 p.Met665Arg mutation shows altered mitochondrial bioenergetics (decreased basal oxygen consumption, ATP synthesis, and membrane potential), reduced Opa1 fusogenic isoforms, and altered mitochondrial network morphology in MEFs. Chloramphenicol treatment (inhibiting mitochondrial protein synthesis) reverses mitochondrial morphology defects, supporting mitochondrial proteotoxicity as disease mechanism.","method":"Knock-in mouse model, MEF mitochondrial respiration (Seahorse), Western blot, mitochondrial morphology analysis, chloramphenicol pharmacological rescue","journal":"Neurobiology of disease","confidence":"High","confidence_rationale":"Tier 2 / Strong — knock-in mouse model with multiple orthogonal mechanistic readouts plus pharmacological rescue experiment","pmids":["30389403"],"is_preprint":false},{"year":2023,"finding":"AFG3L2 (m-AAA protease) directly degrades SLC25A39, a mitochondrial glutathione transporter, through the transporter's matrix loop 1. SLC25A39 also senses mitochondrial iron-sulfur clusters via four matrix cysteine residues that inhibit its degradation by AFG3L2, providing dual regulation of mitochondrial glutathione homeostasis. This was established by Co-IP mass spectrometry and CRISPR KO in mammalian cells.","method":"Co-immunoprecipitation mass spectrometry, CRISPR KO, mutagenesis of degradation signals, glutathione measurement","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — Co-IP MS identification of substrate plus CRISPR KO validation plus mutagenesis of degron and iron-sensing residues, multiple orthogonal methods","pmids":["38157846"],"is_preprint":false},{"year":2025,"finding":"Afg3l2 mediates degradation of Mmadhc (a cobalamin trafficking protein) in hematopoietic stem cells; loss of Afg3l2 leads to Mmadhc accumulation, excessive mitochondrial cobalamin import, elevated adenosylcobalamin, hyperactivation of methylmalonyl-CoA mutase, and increased succinyl-CoA production, impairing HSC maintenance. Mmadhc overexpression phenocopies Afg3l2 deficiency, and Mmadhc knockdown partially rescues HSC function.","method":"Conditional KO mouse, proteomics, Mmadhc overexpression and knockdown rescue experiments, metabolomics, HSC functional assays","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO plus gain-of-function and loss-of-function rescue experiments with metabolic mechanistic dissection, multiple orthogonal methods","pmids":["41411131"],"is_preprint":false},{"year":2025,"finding":"The mitochondrial m-AAA protease AFG3L2 constitutively degrades VISA/MAVS under physiological conditions, thereby negatively regulating RLR-mediated innate antiviral signaling. Physalin F binds to and promotes activation of AFG3L2, which then mediates VISA degradation. AFG3L2 knockdown enhances RLR-mediated innate antiviral signaling.","method":"AFG3L2 knockdown, physalin F binding and activation assays, VISA degradation assays, innate immune signaling readouts in cells and mice","journal":"Pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct knockdown functional experiments and pharmacological activation, single lab, mechanistic link established but full reconstitution not described","pmids":["41599057"],"is_preprint":false},{"year":2025,"finding":"OMA1 cleaves the mitochondrial chaperone DNAJC15, promoting its degradation by the m-AAA protease AFG3L2. Loss of DNAJC15 reduces import of OXPHOS-related proteins via the TIMM23-TIMM17A translocase, limiting OXPHOS biogenesis under mitochondrial dysfunction, linking AFG3L2-mediated proteostasis to mitochondrial protein import regulation.","method":"DNAJC15 degradation assay, AFG3L2 knockout/knockdown, protein import assays, OXPHOS biogenesis analysis","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct biochemical demonstration of AFG3L2-mediated DNAJC15 degradation with functional import readouts, preprint not yet peer-reviewed","pmids":[],"is_preprint":true},{"year":2025,"finding":"AFG3L2 haploinsufficiency (heterozygous truncating variant) can cause axonal Charcot-Marie-Tooth neuropathy; patient fibroblasts show ~50% reduction in AFG3L2 protein, OMA1 hyperactivation, increased OPA1 processing, mitochondrial shortening, and activation of the integrated stress response.","method":"Clinical exome sequencing, patient fibroblast Western blot (AFG3L2, OPA1, OMA1), mitochondrial morphology analysis, integrated stress response markers","journal":"Neurology. Genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct patient fibroblast mechanistic studies with multiple biochemical readouts, single case but multiple orthogonal methods","pmids":["41883704"],"is_preprint":false}],"current_model":"AFG3L2 is an inner mitochondrial membrane AAA+ metalloprotease that forms homo-oligomeric complexes and hetero-oligomeric complexes with paraplegin (SPG7); its cryo-EM structure reveals specialized substrate-engagement features that drive ATP-dependent protein unfolding and degradation with a substrate specificity dominated by P1' residue identity, and its defined substrates include MrpL32 (required for mitochondrial ribosome assembly and protein synthesis), SLC25A39 (a glutathione transporter regulated by iron-sulfur cluster sensing), Mmadhc (controlling cobalamin metabolism), VISA/MAVS (innate immune adaptor), and DNAJC15 (mitochondrial chaperone); AFG3L2 also cleaves and activates SPG7 in a process regulated by tyrosine phosphorylation, and controls OPA1 processing via OMA1 to regulate mitochondrial fusion, with loss of function causing respiratory chain deficiency, mitochondrial fragmentation, defective Ca2+ buffering, and neurodegeneration predominantly affecting Purkinje cells."},"narrative":{"mechanistic_narrative":"AFG3L2 is an inner mitochondrial membrane AAA+ metalloprotease that governs mitochondrial protein quality control and organelle homeostasis [PMID:18337413]. It assembles into homo-oligomeric complexes and into hetero-oligomeric m-AAA protease complexes with paraplegin (SPG7), a capacity for homo-oligomerization that distinguishes it from SPG7 and underlies its broader importance in neurons [PMID:18337413]; AFG3L2 itself cleaves and activates SPG7 upon complex assembly, a step regulated by tyrosine phosphorylation [PMID:24767997]. The cryo-EM structure of its substrate-bound catalytic core reveals specialized features that integrate with conserved AAA+ motifs to drive ATP-dependent substrate translocation, unfolding, and degradation, with peptidase specificity strongly constrained by the P1' residue [PMID:29932645, PMID:31327635]. Through this proteolytic activity AFG3L2 processes MrpL32 to enable mitochondrial ribosome assembly and protein synthesis [PMID:23041622, PMID:29932645] and regulates a defined substrate set including the glutathione transporter SLC25A39 (whose degradation is inhibited by iron-sulfur cluster sensing) [PMID:38157846], the cobalamin-trafficking protein Mmadhc [PMID:41411131], the innate immune adaptor VISA/MAVS [PMID:41599057], and the chaperone DNAJC15. AFG3L2 also controls mitochondrial dynamics by governing OPA1 processing through OMA1, so that loss of function provokes OMA1 hyperactivation, OPA1 short-form accumulation, and mitochondrial fragmentation [PMID:30910913, PMID:22678058]. Disease arises through these axes: proteolytic-domain mutations cause SCA28 via impaired mitochondrial protein synthesis and respiratory chain (complex IV) deficiency [PMID:20208537, PMID:23041622, PMID:30544562], whereas ATPase/AAA-domain mutations cause dominant optic atrophy through OPA1-processing defects and fragmentation [PMID:32219868, PMID:32600459], with haploinsufficiency additionally linked to recessive spastic ataxia-neuropathy and axonal Charcot-Marie-Tooth neuropathy [PMID:22022284, PMID:41883704]. In neurons, AFG3L2 loss converges on respiratory dysfunction, mitochondrial fragmentation, and defective Ca2+ buffering that drives Purkinje cell degeneration [PMID:19625515, PMID:25485680].","teleology":[{"year":1999,"claim":"Establishing that AFG3L2 is a mitochondrial protein co-localizing with paraplegin defined its compartment and first linked it to the same machinery as SPG7.","evidence":"Immunofluorescence and radiation hybrid mapping of a cloned 797-aa protein homologous to yeast Afg3p/Rca1p","pmids":["10395799"],"confidence":"Medium","gaps":["No demonstration of protease activity or substrates","Oligomeric organization not resolved"]},{"year":2008,"claim":"Showing AFG3L2 forms both homo- and hetero-oligomeric m-AAA complexes explained why it has broader, neuron-critical functions than paraplegin.","evidence":"Null and missense mutant mouse models with histology and complex assembly analysis","pmids":["18337413"],"confidence":"High","gaps":["Specific protease substrates not defined","Molecular basis of homo- vs hetero-oligomerization preference unresolved"]},{"year":2009,"claim":"Haploinsufficiency modeling established a mitochondria-mediated pathogenic mechanism for SCA28 centered on respiratory dysfunction and Purkinje cell loss.","evidence":"Afg3l2 haploinsufficient mouse, electron microscopy, ROS measurement, histology","pmids":["19625515"],"confidence":"High","gaps":["Did not pinpoint which substrates drive respiratory failure","Link to Ca2+ handling not yet made"]},{"year":2010,"claim":"Identifying heterozygous proteolytic-domain missense mutations as the cause of SCA28 tied human disease directly to impaired proteolytic and respiratory function.","evidence":"Yeast complementation with respiratory and proteolytic readouts, family sequencing, homology modeling","pmids":["20208537"],"confidence":"High","gaps":["Substrate-level consequences inferred from modeling, not measured","Complex IV deficiency mechanism not yet traced to protein synthesis"]},{"year":2011,"claim":"A homozygous Y616C hypomorph causing spastic ataxia-neuropathy showed that oligomerization defects, not catalytic-site loss alone, can drive disease.","evidence":"Yeast complementation and Blue Native PAGE of patient fibroblasts, whole-exome sequencing","pmids":["22022284"],"confidence":"High","gaps":["Affected substrates not identified","Genotype-phenotype basis for recessive vs dominant inheritance unresolved"]},{"year":2012,"claim":"Conditional and constitutive knockouts identified defective mitochondrial protein synthesis via impaired ribosome assembly as the central causative mechanism of cell-autonomous Purkinje neurodegeneration.","evidence":"Cell-specific and constitutive Afg3l2 KO mice, protein synthesis assays, ribosome assembly analysis, live imaging","pmids":["23041622"],"confidence":"High","gaps":["Direct substrate connecting AFG3L2 to ribosome assembly not yet defined","Relationship to OPA1/dynamics not addressed"]},{"year":2012,"claim":"Linking AFG3L2 loss to reduced mitochondrial Ca2+ uptake via OPA1-dependent fragmentation established the dynamics-Ca2+ axis and separated it from respiratory rescue.","evidence":"Afg3l2 KO MEFs, Ca2+ imaging, permeabilized-cell assays, OPA1 overexpression rescue","pmids":["22678058"],"confidence":"High","gaps":["OPA1 overexpression did not restore respiration, leaving the two defects mechanistically separable but unintegrated","Protease that processes OPA1 not yet identified as OMA1 here"]},{"year":2014,"claim":"Demonstrating that AFG3L2 cleaves and activates SPG7, under phosphorylation control, revealed a regulatory hierarchy within the m-AAA complex.","evidence":"Biochemical processing and phosphorylation assays in cell lines, ROS and ATP measurements","pmids":["24767997"],"confidence":"High","gaps":["Kinase/phosphatase regulating AFG3L2 phosphorylation not identified","Physiological triggers of this regulation unknown"]},{"year":2014,"claim":"Showing that defective mitochondrial Ca2+ buffering drives Purkinje dark degeneration, and that mGluR1/glutamate modulation rescues it, provided a tractable downstream therapeutic axis.","evidence":"Afg3l2 haploinsufficient mice, Ca2+ imaging, genetic mGluR1 silencing, ceftriaxone rescue","pmids":["25485680"],"confidence":"High","gaps":["Rescue addresses downstream Ca2+ handling, not the upstream proteostatic defect","Generalizability beyond Purkinje cells unclear"]},{"year":2018,"claim":"Defining a presequence degron on MrpL32 and profiling P1' specificity gave the first molecular rules for AFG3L2 substrate selection and product length.","evidence":"Solubilized AFG3L2 in vitro assays, MS-based peptidase profiling, fluorogenic peptides, degron mutagenesis","pmids":["29932645"],"confidence":"High","gaps":["Specificity rules derived in vitro; in vivo substrate repertoire incomplete","How the membrane context shapes specificity not addressed"]},{"year":2018,"claim":"Comparing AFG3L2 and YME1L knockdowns assigned AFG3L2 a specific role in complex IV biogenesis and showed combinatorial control of OPA1 processing.","evidence":"siRNA knockdown, Blue Native PAGE, EM, respiratory assays, Western blot","pmids":["30544562"],"confidence":"Medium","gaps":["Single-lab knockdown study","Direct substrates underlying complex IV defect not identified"]},{"year":2018,"claim":"A knock-in M665R mouse confirmed that bioenergetic and OPA1/morphology defects are driven by mitochondrial proteotoxicity reversible by suppressing protein synthesis.","evidence":"Knock-in mouse, Seahorse respirometry, Western blot, chloramphenicol rescue","pmids":["30389403"],"confidence":"High","gaps":["Mechanism linking unfolded protein burden to OPA1 processing not fully resolved here","Neuronal versus fibroblast differences not addressed"]},{"year":2018,"claim":"An AFG3L2 R468C variant with concurrent SPG7 deletion revealed a distinct OPA1-processing/fragmentation mechanism not seen in classic SCA28 cells.","evidence":"Yeast complementation and patient fibroblast OPA1/morphology analysis","pmids":["30252181"],"confidence":"Medium","gaps":["Single patient","Interaction between AFG3L2 mutation and SPG7 loss not fully dissected"]},{"year":2019,"claim":"Demonstrating that altered proteostasis hyperactivates OMA1 to over-process OPA1 connected AFG3L2 protease function to mitochondrial fusion via a defined OMA1-OPA1 axis.","evidence":"Patient fibroblasts, CRISPR KO HEK293T, Afg3l2-/- MEFs, OPA1/OMA1 Western blot, chloramphenicol rescue","pmids":["30910913"],"confidence":"High","gaps":["Signal triggering OMA1 activation downstream of proteostatic stress not molecularly defined","Substrate whose accumulation activates OMA1 unknown"]},{"year":2019,"claim":"The cryo-EM structure of the substrate-bound catalytic core provided the molecular framework for ATP-driven translocation and a structural rationale for disease mutations.","evidence":"Cryo-EM, mutagenesis of disease residues, enzymatic activity assays","pmids":["31327635"],"confidence":"High","gaps":["Structure is of the catalytic core, not the full membrane-embedded complex","Hetero-complex with SPG7 not structurally resolved"]},{"year":2020,"claim":"Showing that ATPase/AAA-domain mutations cause dominant optic atrophy through OPA1 mis-processing established a domain-specific genotype-mechanism distinction from proteolytic-domain SCA28.","evidence":"Targeted NGS/WES, yeast complementation, patient fibroblast OPA1/morphology analysis","pmids":["32219868","32600459"],"confidence":"High","gaps":["Why ATPase-domain defects preferentially affect optic nerve unclear","Quantitative contribution of fragmentation versus respiration not separated"]},{"year":2023,"claim":"Identifying SLC25A39 as a substrate degraded via its matrix loop, with iron-sulfur cluster sensing inhibiting degradation, placed AFG3L2 at the center of glutathione homeostasis regulation.","evidence":"Co-IP mass spectrometry, CRISPR KO, degron/cysteine mutagenesis, glutathione measurement","pmids":["38157846"],"confidence":"High","gaps":["How iron-sulfur status is communicated to the protease mechanistically unresolved","Contribution of this axis to neurodegeneration not tested"]},{"year":2025,"claim":"Defining Mmadhc as an AFG3L2 substrate whose accumulation deranges cobalamin/methylmalonyl-CoA metabolism extended AFG3L2 function to hematopoietic stem cell maintenance.","evidence":"Conditional KO mouse, proteomics, Mmadhc overexpression/knockdown rescue, metabolomics, HSC assays","pmids":["41411131"],"confidence":"High","gaps":["Whether this axis operates in neurons not addressed","Direct cleavage site on Mmadhc not mapped"]},{"year":2025,"claim":"Establishing that AFG3L2 constitutively degrades VISA/MAVS revealed a role in negatively regulating RLR antiviral signaling, expanding its function beyond canonical proteostasis.","evidence":"AFG3L2 knockdown, physalin F binding/activation, VISA degradation and immune signaling readouts in cells and mice","pmids":["41599057"],"confidence":"Medium","gaps":["Full enzymatic reconstitution of VISA degradation not described","Physiological signals that tune this activity unknown"]},{"year":2025,"claim":"Linking OMA1-cleaved DNAJC15 degradation by AFG3L2 to TIMM23-TIMM17A-dependent OXPHOS protein import connected AFG3L2 proteostasis to mitochondrial protein import control.","evidence":"DNAJC15 degradation assay, AFG3L2 KO/KD, protein import and OXPHOS biogenesis analysis (preprint)","pmids":[],"confidence":"Medium","gaps":["Preprint, not peer-reviewed","Physiological conditions activating this OMA1-AFG3L2-DNAJC15 cascade not defined"]},{"year":2025,"claim":"A heterozygous truncating variant causing axonal CMT with OMA1 hyperactivation and integrated stress response activation extended the AFG3L2 haploinsufficiency phenotype spectrum.","evidence":"Clinical exome sequencing, patient fibroblast Western blot, mitochondrial morphology, ISR markers","pmids":["41883704"],"confidence":"Medium","gaps":["Single case","Causal link between ISR activation and neuropathy not established"]},{"year":null,"claim":"How AFG3L2 substrate selection, oligomeric state, and phosphorylation are coordinated in vivo to triage between protein quality control, OPA1/OMA1-mediated dynamics, metabolic regulation, and immune signaling remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model integrating the multiple substrate axes","Structure of the full membrane-embedded hetero-complex with SPG7 not determined","Upstream signals controlling AFG3L2 phosphorylation and activation unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[9,12,17,18,20]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[3,9,17]},{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[12,14]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[0,1]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[5,9,17,18]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[6,13,14]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[3,4,14,21]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[19]}],"complexes":["m-AAA protease"],"partners":["SPG7"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9Y4W6","full_name":"Mitochondrial inner membrane m-AAA protease component AFG3L2","aliases":["AFG3-like protein 2","Paraplegin-like protein"],"length_aa":797,"mass_kda":88.6,"function":"Catalytic component of the m-AAA protease, a protease that plays a key role in proteostasis of inner mitochondrial membrane proteins, and which is essential for axonal and neuron development (PubMed:19748354, PubMed:28396416, PubMed:29932645, PubMed:30683687, PubMed:31327635, PubMed:37917749, PubMed:38157846). AFG3L2 possesses both ATPase and protease activities: the ATPase activity is required to unfold substrates, threading them into the internal proteolytic cavity for hydrolysis into small peptide fragments (PubMed:19748354, PubMed:31327635). The m-AAA protease carries out quality control in the inner membrane of the mitochondria by mediating degradation of mistranslated or misfolded polypeptides (PubMed:26504172, PubMed:30683687, PubMed:34718584). The m-AAA protease complex also promotes the processing and maturation of mitochondrial proteins, such as MRPL32/bL32m, PINK1 and SP7 (PubMed:22354088, PubMed:29932645, PubMed:30252181). Mediates protein maturation of the mitochondrial ribosomal subunit MRPL32/bL32m by catalyzing the cleavage of the presequence of MRPL32/bL32m prior to assembly into the mitochondrial ribosome (PubMed:29932645). Required for SPG7 maturation into its active mature form after SPG7 cleavage by mitochondrial-processing peptidase (MPP) (PubMed:30252181). Required for the maturation of PINK1 into its 52kDa mature form after its cleavage by mitochondrial-processing peptidase (MPP) (PubMed:22354088). Acts as a regulator of calcium in neurons by mediating degradation of SMDT1/EMRE before its assembly with the uniporter complex, limiting the availability of SMDT1/EMRE for MCU assembly and promoting efficient assembly of gatekeeper subunits with MCU (PubMed:27642048, PubMed:28396416). Promotes the proteolytic degradation of GHITM upon hyperpolarization of mitochondria: progressive GHITM degradation leads to respiratory complex I degradation and broad reshaping of the mitochondrial proteome by AFG3L2 (PubMed:35912435). Also acts as a regulator of mitochondrial glutathione homeostasis by mediating cleavage and degradation of SLC25A39 (PubMed:37917749, PubMed:38157846). SLC25A39 cleavage is prevented when SLC25A39 binds iron-sulfur (PubMed:37917749, PubMed:38157846). Involved in the regulation of OMA1-dependent processing of OPA1 (PubMed:17615298, PubMed:29545505, PubMed:30252181, PubMed:30683687, PubMed:32600459). May act by mediating processing of OMA1 precursor, participating in OMA1 maturation (PubMed:29545505)","subcellular_location":"Mitochondrion inner membrane","url":"https://www.uniprot.org/uniprotkb/Q9Y4W6/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/AFG3L2","classification":"Common Essential","n_dependent_lines":1177,"n_total_lines":1208,"dependency_fraction":0.9743377483443708},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/AFG3L2","total_profiled":1310},"omim":[{"mim_id":"618977","title":"OPTIC ATROPHY 12; OPA12","url":"https://www.omim.org/entry/618977"},{"mim_id":"617267","title":"MATRIX AAA PEPTIDASE-INTERACTING PROTEIN 1; MAIP1","url":"https://www.omim.org/entry/617267"},{"mim_id":"615898","title":"NADH DEHYDROGENASE (UBIQUINONE) COMPLEX I, ASSEMBLY FACTOR 7; NDUFAF7","url":"https://www.omim.org/entry/615898"},{"mim_id":"615588","title":"SINGLE-PASS MEMBRANE PROTEIN WITH ASPARTATE-RICH TAIL 1; SMDT1","url":"https://www.omim.org/entry/615588"},{"mim_id":"614487","title":"SPASTIC ATAXIA 5, AUTOSOMAL RECESSIVE; SPAX5","url":"https://www.omim.org/entry/614487"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Mitochondria","reliability":"Enhanced"},{"location":"Perinuclear theca","reliability":"Additional"},{"location":"Calyx","reliability":"Additional"},{"location":"Connecting piece","reliability":"Additional"},{"location":"Mid piece","reliability":"Additional"},{"location":"Principal piece","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"skeletal muscle","ntpm":96.6},{"tissue":"tongue","ntpm":92.6}],"url":"https://www.proteinatlas.org/search/AFG3L2"},"hgnc":{"alias_symbol":["SPAX5"],"prev_symbol":["SCA28"]},"alphafold":{"accession":"Q9Y4W6","domains":[{"cath_id":"3.40.1690.20","chopping":"146-268","consensus_level":"medium","plddt":79.8761,"start":146,"end":268},{"cath_id":"3.40.50.300","chopping":"298-475","consensus_level":"high","plddt":87.1812,"start":298,"end":475},{"cath_id":"1.10.8.60","chopping":"481-555","consensus_level":"high","plddt":93.7904,"start":481,"end":555},{"cath_id":"1.20.58.760","chopping":"566-680_690-760","consensus_level":"high","plddt":92.7572,"start":566,"end":760}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y4W6","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y4W6-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y4W6-F1-predicted_aligned_error_v6.png","plddt_mean":76.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=AFG3L2","jax_strain_url":"https://www.jax.org/strain/search?query=AFG3L2"},"sequence":{"accession":"Q9Y4W6","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9Y4W6.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9Y4W6/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y4W6"}},"corpus_meta":[{"pmid":"20208537","id":"PMC_20208537","title":"Mutations 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Loss of AFG3L2 in mice causes marked impairment of axonal development with delayed myelination and poor axonal radial growth, leading to lethality at P16.\",\n      \"method\": \"Null and spontaneous missense mutant mouse models, histological analysis, immunofluorescence\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — two independent murine models (null and missense) with defined cellular phenotypes and molecular mechanism (homo- vs hetero-oligomerization) replicated across models\",\n      \"pmids\": [\"18337413\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Haploinsufficiency of Afg3l2 in mice causes respiratory chain dysfunction, increased reactive oxygen species production, and dark degeneration of Purkinje cells, establishing a mitochondria-mediated pathogenic mechanism for SCA28.\",\n      \"method\": \"Afg3l2 haploinsufficient mouse model, electron microscopy, ROS measurement, histology\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetically defined haploinsufficient mouse model with multiple orthogonal mechanistic readouts (ROS, respiratory chain, cell degeneration)\",\n      \"pmids\": [\"19625515\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Heterozygous missense mutations in AFG3L2 cause SCA28; m-AAA-deficient yeast expressing mutated human AFG3L2 homocomplex show respiratory deficiency, proteolytic impairment, and deficiency of respiratory chain complex IV. Mutations cluster in the proteolytic domain and are predicted by homology modeling to affect substrate handling.\",\n      \"method\": \"Yeast complementation assay, respiratory growth assay, structure homology modeling, Sanger sequencing in families\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — yeast functional reconstitution with proteolytic and respiratory readouts, replicated in multiple independent families, supported by structural modeling\",\n      \"pmids\": [\"20208537\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"A homozygous AFG3L2 Y616C mutation causes recessive spastic ataxia-neuropathy; the Y616C variant is hypomorphic, exhibiting oligomerization defects both in yeast complementation assays and in patient fibroblasts — specifically, formation of AFG3L2(Y616C) homo-complexes and hetero-complexes with paraplegin is impaired.\",\n      \"method\": \"Yeast complementation assay, Blue Native PAGE in patient fibroblasts, whole-exome sequencing\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — yeast complementation plus patient fibroblast biochemistry, two orthogonal methods demonstrating oligomerization defect\",\n      \"pmids\": [\"22022284\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Conditional knockout of Afg3l2 in mouse Purkinje cells causes cell-autonomous neurodegeneration with early mitochondrial fragmentation and altered distribution in the dendritic tree. Constitutive knockout reveals a decreased rate of mitochondrial protein synthesis associated with impaired mitochondrial ribosome assembly, establishing defective mitochondrial protein synthesis as a central causative mechanism.\",\n      \"method\": \"Conditional and constitutive Afg3l2 knockout mouse models, live imaging, electron microscopy, mitochondrial protein synthesis assay, ribosome assembly analysis\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — cell-autonomous KO with multiple orthogonal mechanistic readouts (protein synthesis rate, ribosome assembly, mitochondrial morphology) across two mouse models\",\n      \"pmids\": [\"23041622\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Loss of AFG3L2 in mouse embryonic fibroblasts reduces mitochondrial Ca2+ uptake capacity, caused by fragmentation of the mitochondrial network secondary to respiratory dysfunction and consequent OPA1 processing. Overexpression of OPA1 in Afg3l2-/- MEFs rescues impaired mitochondrial Ca2+ buffering but fails to restore respiration.\",\n      \"method\": \"Afg3l2 KO MEFs, mitochondrial Ca2+ imaging, permeabilized cell assays, OPA1 overexpression rescue experiment\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Ca2+ imaging, rescue overexpression, permeabilized cells) establishing the OPA1-fragmentation-Ca2+ axis\",\n      \"pmids\": [\"22678058\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"AFG3L2 regulates SPG7 (paraplegin) processing: SPG7 is cleaved and activated by AFG3L2 upon assembly into the mAAA protease complex. This processing is regulated by tyrosine phosphorylation of AFG3L2; the SPG7 Q688 variant bypasses this regulation and constitutively activates SPG7 mAAA protease, resulting in elevated ATP production and ROS.\",\n      \"method\": \"Biochemical processing assays in cell lines, phosphorylation studies, mitochondrial ROS and ATP measurements\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct biochemical demonstration of AFG3L2-mediated SPG7 cleavage and phosphorylation-dependent regulation with multiple functional readouts\",\n      \"pmids\": [\"24767997\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"In SCA28 mice, mitochondria in Afg3l2-deficient Purkinje cells fail to buffer evoked Ca2+ peaks, resulting in enhanced cytoplasmic Ca2+ concentrations triggering dark degeneration. This Ca2+ handling defect results from negative synergism between mitochondrial depolarization and altered organelle trafficking to dendrites. Partial genetic silencing of mGluR1 or ceftriaxone-mediated glutamate clearance rescued the ataxic phenotype.\",\n      \"method\": \"Afg3l2 haploinsufficient mouse model, Ca2+ imaging in cultured PCs, genetic mGluR1 silencing, pharmacological rescue with ceftriaxone\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct Ca2+ imaging with mechanistic dissection plus two independent rescue strategies (genetic and pharmacological) in vivo and in vitro\",\n      \"pmids\": [\"25485680\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"AFG3L2 contains multiple substrate specificity mechanisms: conserved residues in the presequence of mitochondrial ribosomal protein MrpL32 serve as a degron targeting it for processing by AFG3L2. Peptidase specificity profiling using mass spectrometry reveals constrained product lengths with strong preference for hydrophobic and small polar residues at the P1' position.\",\n      \"method\": \"Solubilized AFG3L2 in vitro assay, mass spectrometry-based peptidase specificity profiling, fluorogenic reporter peptides, mutagenesis of degron sequences\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic reconstitution with mutagenesis, MS-based specificity profiling, and functional validation with multiple substrates, single lab\",\n      \"pmids\": [\"29932645\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Loss of AFG3L2 and YME1L, individually and in combination, results in diminished cell proliferation, mitochondrial reticulum fragmentation, altered cristae morphogenesis, and defective respiratory chain biogenesis. AFG3L2 knockdown specifically impairs assembly and function of complex IV, while YME1L loss impairs complex I. Double knockdown causes marked OPA1 short form accumulation.\",\n      \"method\": \"siRNA knockdown, Blue Native PAGE, electron microscopy, respiratory chain activity assays, Western blot\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple knockdown conditions with orthogonal readouts, single lab\",\n      \"pmids\": [\"30544562\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In a patient carrying AFG3L2 p.R468C with concurrent SPG7 deletion, patient fibroblasts show abnormal OPA1 processing with mitochondrial network fragmentation — a phenotype not seen in SCA28 or SPG7 patients' cells alone. Yeast functional analysis confirmed pathogenicity of AFG3L2 p.R468C and revealed its distinct pathogenic mechanism from classic SCA28 mutations.\",\n      \"method\": \"Yeast complementation assay, patient fibroblast OPA1 processing analysis, mitochondrial morphology assessment\",\n      \"journal\": \"Human mutation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast complementation plus patient cell biochemistry, single lab, two methods\",\n      \"pmids\": [\"30252181\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Cryo-EM structure of the substrate-bound catalytic core of human AFG3L2 reveals multiple specialized structural features that integrate with conserved AAA+ motifs for ATP-dependent substrate translocation, unfolding, and degradation. Disease-relevant mutations localize to these unique structural features and distinctly influence AFG3L2 activity and stability.\",\n      \"method\": \"Cryo-electron microscopy, mutagenesis of disease-associated residues, enzymatic activity assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure with functional validation by mutagenesis and activity assays, providing molecular basis for disease mutations\",\n      \"pmids\": [\"31327635\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SCA28 patient fibroblasts carrying proteolytic domain missense mutations show inefficient mitochondrial fusion caused by increased OPA1 processing operated by hyperactivated OMA1. Altered mitochondrial proteostasis triggers OMA1 activation, and pharmacological attenuation of mitochondrial protein synthesis stabilizes OMA1 and OPA1 long forms, rescuing mitochondrial fusion efficiency.\",\n      \"method\": \"Patient fibroblasts, CRISPR/Cas9 AFG3L2 KO HEK293T cells, Afg3l2-/- MEFs, OPA1/OMA1 Western blot, Blue Native PAGE, mitochondrial Ca2+ measurement, chloramphenicol treatment rescue\",\n      \"journal\": \"Journal of medical genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple patient cell lines plus isogenic KO models, pharmacological rescue, and mechanistic dissection of OMA1-OPA1 axis, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"30910913\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"AFG3L2 mutations in the ATPase domain (distinct from the proteolytic domain mutations causing SCA28) cause dominant optic atrophy through a different mechanism: patient fibroblasts show abnormal OPA1 processing with accumulation of fission-inducing short forms and mitochondrial network fragmentation. Pathogenicity confirmed in yeast.\",\n      \"method\": \"Targeted NGS/WES, yeast complementation assay, patient fibroblast OPA1 processing analysis, mitochondrial morphology assessment\",\n      \"journal\": \"Annals of neurology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — yeast functional validation plus patient fibroblast biochemistry, mechanistic distinction from SCA28 established with multiple methods\",\n      \"pmids\": [\"32219868\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"A novel AFG3L2 mutation (p.G337E) close to the AAA domain strongly destabilizes OPA1 long isoforms via OMA1 hyperactivation, leading to mitochondrial fragmentation in patient fibroblasts — a mechanism similar to ATPase-domain DOA mutations but distinct from proteolytic-domain SCA28 mutations.\",\n      \"method\": \"Patient fibroblast functional studies, OPA1/OMA1 Western blot, mitochondrial morphology assessment\",\n      \"journal\": \"Acta neuropathologica communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct patient fibroblast experiments with OMA1/OPA1 biochemical readouts, single lab, two orthogonal methods\",\n      \"pmids\": [\"32600459\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"A knock-in mouse model carrying the SCA28 patient-derived Afg3l2 p.Met665Arg mutation shows altered mitochondrial bioenergetics (decreased basal oxygen consumption, ATP synthesis, and membrane potential), reduced Opa1 fusogenic isoforms, and altered mitochondrial network morphology in MEFs. Chloramphenicol treatment (inhibiting mitochondrial protein synthesis) reverses mitochondrial morphology defects, supporting mitochondrial proteotoxicity as disease mechanism.\",\n      \"method\": \"Knock-in mouse model, MEF mitochondrial respiration (Seahorse), Western blot, mitochondrial morphology analysis, chloramphenicol pharmacological rescue\",\n      \"journal\": \"Neurobiology of disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — knock-in mouse model with multiple orthogonal mechanistic readouts plus pharmacological rescue experiment\",\n      \"pmids\": [\"30389403\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"AFG3L2 (m-AAA protease) directly degrades SLC25A39, a mitochondrial glutathione transporter, through the transporter's matrix loop 1. SLC25A39 also senses mitochondrial iron-sulfur clusters via four matrix cysteine residues that inhibit its degradation by AFG3L2, providing dual regulation of mitochondrial glutathione homeostasis. This was established by Co-IP mass spectrometry and CRISPR KO in mammalian cells.\",\n      \"method\": \"Co-immunoprecipitation mass spectrometry, CRISPR KO, mutagenesis of degradation signals, glutathione measurement\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — Co-IP MS identification of substrate plus CRISPR KO validation plus mutagenesis of degron and iron-sensing residues, multiple orthogonal methods\",\n      \"pmids\": [\"38157846\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Afg3l2 mediates degradation of Mmadhc (a cobalamin trafficking protein) in hematopoietic stem cells; loss of Afg3l2 leads to Mmadhc accumulation, excessive mitochondrial cobalamin import, elevated adenosylcobalamin, hyperactivation of methylmalonyl-CoA mutase, and increased succinyl-CoA production, impairing HSC maintenance. Mmadhc overexpression phenocopies Afg3l2 deficiency, and Mmadhc knockdown partially rescues HSC function.\",\n      \"method\": \"Conditional KO mouse, proteomics, Mmadhc overexpression and knockdown rescue experiments, metabolomics, HSC functional assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO plus gain-of-function and loss-of-function rescue experiments with metabolic mechanistic dissection, multiple orthogonal methods\",\n      \"pmids\": [\"41411131\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The mitochondrial m-AAA protease AFG3L2 constitutively degrades VISA/MAVS under physiological conditions, thereby negatively regulating RLR-mediated innate antiviral signaling. Physalin F binds to and promotes activation of AFG3L2, which then mediates VISA degradation. AFG3L2 knockdown enhances RLR-mediated innate antiviral signaling.\",\n      \"method\": \"AFG3L2 knockdown, physalin F binding and activation assays, VISA degradation assays, innate immune signaling readouts in cells and mice\",\n      \"journal\": \"Pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct knockdown functional experiments and pharmacological activation, single lab, mechanistic link established but full reconstitution not described\",\n      \"pmids\": [\"41599057\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"OMA1 cleaves the mitochondrial chaperone DNAJC15, promoting its degradation by the m-AAA protease AFG3L2. Loss of DNAJC15 reduces import of OXPHOS-related proteins via the TIMM23-TIMM17A translocase, limiting OXPHOS biogenesis under mitochondrial dysfunction, linking AFG3L2-mediated proteostasis to mitochondrial protein import regulation.\",\n      \"method\": \"DNAJC15 degradation assay, AFG3L2 knockout/knockdown, protein import assays, OXPHOS biogenesis analysis\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct biochemical demonstration of AFG3L2-mediated DNAJC15 degradation with functional import readouts, preprint not yet peer-reviewed\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"AFG3L2 haploinsufficiency (heterozygous truncating variant) can cause axonal Charcot-Marie-Tooth neuropathy; patient fibroblasts show ~50% reduction in AFG3L2 protein, OMA1 hyperactivation, increased OPA1 processing, mitochondrial shortening, and activation of the integrated stress response.\",\n      \"method\": \"Clinical exome sequencing, patient fibroblast Western blot (AFG3L2, OPA1, OMA1), mitochondrial morphology analysis, integrated stress response markers\",\n      \"journal\": \"Neurology. Genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct patient fibroblast mechanistic studies with multiple biochemical readouts, single case but multiple orthogonal methods\",\n      \"pmids\": [\"41883704\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"AFG3L2 is an inner mitochondrial membrane AAA+ metalloprotease that forms homo-oligomeric complexes and hetero-oligomeric complexes with paraplegin (SPG7); its cryo-EM structure reveals specialized substrate-engagement features that drive ATP-dependent protein unfolding and degradation with a substrate specificity dominated by P1' residue identity, and its defined substrates include MrpL32 (required for mitochondrial ribosome assembly and protein synthesis), SLC25A39 (a glutathione transporter regulated by iron-sulfur cluster sensing), Mmadhc (controlling cobalamin metabolism), VISA/MAVS (innate immune adaptor), and DNAJC15 (mitochondrial chaperone); AFG3L2 also cleaves and activates SPG7 in a process regulated by tyrosine phosphorylation, and controls OPA1 processing via OMA1 to regulate mitochondrial fusion, with loss of function causing respiratory chain deficiency, mitochondrial fragmentation, defective Ca2+ buffering, and neurodegeneration predominantly affecting Purkinje cells.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"AFG3L2 is an inner mitochondrial membrane AAA+ metalloprotease that governs mitochondrial protein quality control and organelle homeostasis [#1]. It assembles into homo-oligomeric complexes and into hetero-oligomeric m-AAA protease complexes with paraplegin (SPG7), a capacity for homo-oligomerization that distinguishes it from SPG7 and underlies its broader importance in neurons [#1]; AFG3L2 itself cleaves and activates SPG7 upon complex assembly, a step regulated by tyrosine phosphorylation [#7]. The cryo-EM structure of its substrate-bound catalytic core reveals specialized features that integrate with conserved AAA+ motifs to drive ATP-dependent substrate translocation, unfolding, and degradation, with peptidase specificity strongly constrained by the P1' residue [#9, #12]. Through this proteolytic activity AFG3L2 processes MrpL32 to enable mitochondrial ribosome assembly and protein synthesis [#5, #9] and regulates a defined substrate set including the glutathione transporter SLC25A39 (whose degradation is inhibited by iron-sulfur cluster sensing) [#17], the cobalamin-trafficking protein Mmadhc [#18], the innate immune adaptor VISA/MAVS [#19], and the chaperone DNAJC15 [#20]. AFG3L2 also controls mitochondrial dynamics by governing OPA1 processing through OMA1, so that loss of function provokes OMA1 hyperactivation, OPA1 short-form accumulation, and mitochondrial fragmentation [#13, #6]. Disease arises through these axes: proteolytic-domain mutations cause SCA28 via impaired mitochondrial protein synthesis and respiratory chain (complex IV) deficiency [#3, #5, #10], whereas ATPase/AAA-domain mutations cause dominant optic atrophy through OPA1-processing defects and fragmentation [#14, #15], with haploinsufficiency additionally linked to recessive spastic ataxia-neuropathy and axonal Charcot-Marie-Tooth neuropathy [#4, #21]. In neurons, AFG3L2 loss converges on respiratory dysfunction, mitochondrial fragmentation, and defective Ca2+ buffering that drives Purkinje cell degeneration [#2, #8].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Establishing that AFG3L2 is a mitochondrial protein co-localizing with paraplegin defined its compartment and first linked it to the same machinery as SPG7.\",\n      \"evidence\": \"Immunofluorescence and radiation hybrid mapping of a cloned 797-aa protein homologous to yeast Afg3p/Rca1p\",\n      \"pmids\": [\"10395799\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No demonstration of protease activity or substrates\", \"Oligomeric organization not resolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Showing AFG3L2 forms both homo- and hetero-oligomeric m-AAA complexes explained why it has broader, neuron-critical functions than paraplegin.\",\n      \"evidence\": \"Null and missense mutant mouse models with histology and complex assembly analysis\",\n      \"pmids\": [\"18337413\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific protease substrates not defined\", \"Molecular basis of homo- vs hetero-oligomerization preference unresolved\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Haploinsufficiency modeling established a mitochondria-mediated pathogenic mechanism for SCA28 centered on respiratory dysfunction and Purkinje cell loss.\",\n      \"evidence\": \"Afg3l2 haploinsufficient mouse, electron microscopy, ROS measurement, histology\",\n      \"pmids\": [\"19625515\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not pinpoint which substrates drive respiratory failure\", \"Link to Ca2+ handling not yet made\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Identifying heterozygous proteolytic-domain missense mutations as the cause of SCA28 tied human disease directly to impaired proteolytic and respiratory function.\",\n      \"evidence\": \"Yeast complementation with respiratory and proteolytic readouts, family sequencing, homology modeling\",\n      \"pmids\": [\"20208537\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Substrate-level consequences inferred from modeling, not measured\", \"Complex IV deficiency mechanism not yet traced to protein synthesis\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"A homozygous Y616C hypomorph causing spastic ataxia-neuropathy showed that oligomerization defects, not catalytic-site loss alone, can drive disease.\",\n      \"evidence\": \"Yeast complementation and Blue Native PAGE of patient fibroblasts, whole-exome sequencing\",\n      \"pmids\": [\"22022284\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Affected substrates not identified\", \"Genotype-phenotype basis for recessive vs dominant inheritance unresolved\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Conditional and constitutive knockouts identified defective mitochondrial protein synthesis via impaired ribosome assembly as the central causative mechanism of cell-autonomous Purkinje neurodegeneration.\",\n      \"evidence\": \"Cell-specific and constitutive Afg3l2 KO mice, protein synthesis assays, ribosome assembly analysis, live imaging\",\n      \"pmids\": [\"23041622\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct substrate connecting AFG3L2 to ribosome assembly not yet defined\", \"Relationship to OPA1/dynamics not addressed\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Linking AFG3L2 loss to reduced mitochondrial Ca2+ uptake via OPA1-dependent fragmentation established the dynamics-Ca2+ axis and separated it from respiratory rescue.\",\n      \"evidence\": \"Afg3l2 KO MEFs, Ca2+ imaging, permeabilized-cell assays, OPA1 overexpression rescue\",\n      \"pmids\": [\"22678058\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"OPA1 overexpression did not restore respiration, leaving the two defects mechanistically separable but unintegrated\", \"Protease that processes OPA1 not yet identified as OMA1 here\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Demonstrating that AFG3L2 cleaves and activates SPG7, under phosphorylation control, revealed a regulatory hierarchy within the m-AAA complex.\",\n      \"evidence\": \"Biochemical processing and phosphorylation assays in cell lines, ROS and ATP measurements\",\n      \"pmids\": [\"24767997\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase/phosphatase regulating AFG3L2 phosphorylation not identified\", \"Physiological triggers of this regulation unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Showing that defective mitochondrial Ca2+ buffering drives Purkinje dark degeneration, and that mGluR1/glutamate modulation rescues it, provided a tractable downstream therapeutic axis.\",\n      \"evidence\": \"Afg3l2 haploinsufficient mice, Ca2+ imaging, genetic mGluR1 silencing, ceftriaxone rescue\",\n      \"pmids\": [\"25485680\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Rescue addresses downstream Ca2+ handling, not the upstream proteostatic defect\", \"Generalizability beyond Purkinje cells unclear\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defining a presequence degron on MrpL32 and profiling P1' specificity gave the first molecular rules for AFG3L2 substrate selection and product length.\",\n      \"evidence\": \"Solubilized AFG3L2 in vitro assays, MS-based peptidase profiling, fluorogenic peptides, degron mutagenesis\",\n      \"pmids\": [\"29932645\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specificity rules derived in vitro; in vivo substrate repertoire incomplete\", \"How the membrane context shapes specificity not addressed\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Comparing AFG3L2 and YME1L knockdowns assigned AFG3L2 a specific role in complex IV biogenesis and showed combinatorial control of OPA1 processing.\",\n      \"evidence\": \"siRNA knockdown, Blue Native PAGE, EM, respiratory assays, Western blot\",\n      \"pmids\": [\"30544562\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab knockdown study\", \"Direct substrates underlying complex IV defect not identified\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"A knock-in M665R mouse confirmed that bioenergetic and OPA1/morphology defects are driven by mitochondrial proteotoxicity reversible by suppressing protein synthesis.\",\n      \"evidence\": \"Knock-in mouse, Seahorse respirometry, Western blot, chloramphenicol rescue\",\n      \"pmids\": [\"30389403\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking unfolded protein burden to OPA1 processing not fully resolved here\", \"Neuronal versus fibroblast differences not addressed\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"An AFG3L2 R468C variant with concurrent SPG7 deletion revealed a distinct OPA1-processing/fragmentation mechanism not seen in classic SCA28 cells.\",\n      \"evidence\": \"Yeast complementation and patient fibroblast OPA1/morphology analysis\",\n      \"pmids\": [\"30252181\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single patient\", \"Interaction between AFG3L2 mutation and SPG7 loss not fully dissected\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Demonstrating that altered proteostasis hyperactivates OMA1 to over-process OPA1 connected AFG3L2 protease function to mitochondrial fusion via a defined OMA1-OPA1 axis.\",\n      \"evidence\": \"Patient fibroblasts, CRISPR KO HEK293T, Afg3l2-/- MEFs, OPA1/OMA1 Western blot, chloramphenicol rescue\",\n      \"pmids\": [\"30910913\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Signal triggering OMA1 activation downstream of proteostatic stress not molecularly defined\", \"Substrate whose accumulation activates OMA1 unknown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"The cryo-EM structure of the substrate-bound catalytic core provided the molecular framework for ATP-driven translocation and a structural rationale for disease mutations.\",\n      \"evidence\": \"Cryo-EM, mutagenesis of disease residues, enzymatic activity assays\",\n      \"pmids\": [\"31327635\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure is of the catalytic core, not the full membrane-embedded complex\", \"Hetero-complex with SPG7 not structurally resolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Showing that ATPase/AAA-domain mutations cause dominant optic atrophy through OPA1 mis-processing established a domain-specific genotype-mechanism distinction from proteolytic-domain SCA28.\",\n      \"evidence\": \"Targeted NGS/WES, yeast complementation, patient fibroblast OPA1/morphology analysis\",\n      \"pmids\": [\"32219868\", \"32600459\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why ATPase-domain defects preferentially affect optic nerve unclear\", \"Quantitative contribution of fragmentation versus respiration not separated\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identifying SLC25A39 as a substrate degraded via its matrix loop, with iron-sulfur cluster sensing inhibiting degradation, placed AFG3L2 at the center of glutathione homeostasis regulation.\",\n      \"evidence\": \"Co-IP mass spectrometry, CRISPR KO, degron/cysteine mutagenesis, glutathione measurement\",\n      \"pmids\": [\"38157846\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How iron-sulfur status is communicated to the protease mechanistically unresolved\", \"Contribution of this axis to neurodegeneration not tested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defining Mmadhc as an AFG3L2 substrate whose accumulation deranges cobalamin/methylmalonyl-CoA metabolism extended AFG3L2 function to hematopoietic stem cell maintenance.\",\n      \"evidence\": \"Conditional KO mouse, proteomics, Mmadhc overexpression/knockdown rescue, metabolomics, HSC assays\",\n      \"pmids\": [\"41411131\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this axis operates in neurons not addressed\", \"Direct cleavage site on Mmadhc not mapped\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Establishing that AFG3L2 constitutively degrades VISA/MAVS revealed a role in negatively regulating RLR antiviral signaling, expanding its function beyond canonical proteostasis.\",\n      \"evidence\": \"AFG3L2 knockdown, physalin F binding/activation, VISA degradation and immune signaling readouts in cells and mice\",\n      \"pmids\": [\"41599057\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Full enzymatic reconstitution of VISA degradation not described\", \"Physiological signals that tune this activity unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Linking OMA1-cleaved DNAJC15 degradation by AFG3L2 to TIMM23-TIMM17A-dependent OXPHOS protein import connected AFG3L2 proteostasis to mitochondrial protein import control.\",\n      \"evidence\": \"DNAJC15 degradation assay, AFG3L2 KO/KD, protein import and OXPHOS biogenesis analysis (preprint)\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, not peer-reviewed\", \"Physiological conditions activating this OMA1-AFG3L2-DNAJC15 cascade not defined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"A heterozygous truncating variant causing axonal CMT with OMA1 hyperactivation and integrated stress response activation extended the AFG3L2 haploinsufficiency phenotype spectrum.\",\n      \"evidence\": \"Clinical exome sequencing, patient fibroblast Western blot, mitochondrial morphology, ISR markers\",\n      \"pmids\": [\"41883704\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single case\", \"Causal link between ISR activation and neuropathy not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How AFG3L2 substrate selection, oligomeric state, and phosphorylation are coordinated in vivo to triage between protein quality control, OPA1/OMA1-mediated dynamics, metabolic regulation, and immune signaling remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model integrating the multiple substrate axes\", \"Structure of the full membrane-embedded hetero-complex with SPG7 not determined\", \"Upstream signals controlling AFG3L2 phosphorylation and activation unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [9, 12, 17, 18, 20]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [3, 9, 17]},\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [12, 14]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0005743\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [5, 9, 17, 18]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [6, 13, 14]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [3, 4, 14, 21]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [19]}\n    ],\n    \"complexes\": [\"m-AAA protease\"],\n    \"partners\": [\"SPG7\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}