ACAD9

id: Q9H845
gene_symbol: ACAD9
product_type: PROTEIN
status: COMPLETE
taxon:
  id: NCBITaxon:9606
  label: Homo sapiens
description: >-
  ACAD9 (Complex I assembly factor ACAD9, mitochondrial) is a dual-function protein that
  serves as both a mitochondrial respiratory chain Complex I (CI) assembly factor and an
  acyl-CoA dehydrogenase enzyme. Its primary physiological role is as a core component of
  the mitochondrial complex I intermediate assembly (MCIA) complex, where it partners with
  ECSIT and NDUFAF1 to mediate assembly of the ND2 membrane-arm module of CI. ECSIT binding
  triggers a conformational change and deflavination of ACAD9, switching it from an FAO enzyme
  to a CI assembly factor. As a secondary/moonlighting function, ACAD9 retains FAD-dependent
  acyl-CoA dehydrogenase activity toward long-chain (and some medium-chain) fatty acyl-CoA
  substrates (EC 1.3.8.7, 1.3.8.8), catalyzing the first step of mitochondrial fatty acid
  beta-oxidation. Disease-causing mutations in ACAD9 result in isolated CI deficiency (MC1DN20),
  not primary fatty acid oxidation defects, underscoring the primacy of its assembly role.
  ACAD9 is a homodimer localized to the matrix side of the mitochondrial inner membrane.
existing_annotations:
# --- Annotation 1: acyl-CoA dehydrogenase activity (IBA) ---
- term:
    id: GO:0003995
    label: acyl-CoA dehydrogenase activity
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    summary: >-
      ACAD9 is a bona fide acyl-CoA dehydrogenase, belonging to the ACAD family and retaining
      catalytic activity for the alpha,beta-dehydrogenation of fatty acyl-CoA substrates. The IBA
      annotation is phylogenetically inferred and well-supported by direct biochemical data from
      multiple publications (PMID:16020546, PMID:34646991, PMID:38086790). This is an appropriate
      level of specificity for the IBA evidence code.
    action: ACCEPT
    reason: >-
      ACAD9 has experimentally demonstrated acyl-CoA dehydrogenase activity. Purified ACAD9 shows
      dehydrogenation activity of 83 min-1 with palmitoyl-CoA as substrate (PMID:34646991). The
      IBA annotation at the general acyl-CoA dehydrogenase level is appropriate given phylogenetic
      support across the ACAD family.
    supported_by:
      - reference_id: PMID:34646991
        supporting_text: "As purified, the wild-type ACAD9 protein has a dehydrogenation activity of 83 min-1, attributable to the FAD-containing holo-form of the protein"
      - reference_id: PMID:16020546
        supporting_text: "Purified mature ACAD-9 had maximal activity with long-chain unsaturated acyl-CoAs as substrates (C16:1-, C18:1-, C18:2-, C22:6-CoA)"

# --- Annotation 2: acyl-CoA dehydrogenase activity (IEA, InterPro) ---
- term:
    id: GO:0003995
    label: acyl-CoA dehydrogenase activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  review:
    summary: >-
      IEA annotation based on InterPro domain mapping. ACAD9 contains the acyl-CoA dehydrogenase
      active site signature (IPR006089) which correctly predicts this enzymatic activity. Consistent
      with direct experimental evidence.
    action: ACCEPT
    reason: >-
      The InterPro-based IEA annotation is correct and consistent with experimental data showing
      ACAD9 possesses acyl-CoA dehydrogenase activity (PMID:16020546, PMID:34646991). This is
      a broader annotation that is subsumed by the IBA and IDA annotations but not incorrect.
    supported_by:
      - reference_id: PMID:16020546
        supporting_text: "Purified mature ACAD-9 had maximal activity with long-chain unsaturated acyl-CoAs as substrates"

# --- Annotation 3: long-chain fatty acyl-CoA dehydrogenase activity (IEA, ARBA) ---
- term:
    id: GO:0004466
    label: long-chain fatty acyl-CoA dehydrogenase activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  review:
    summary: >-
      IEA annotation from ARBA/Rhea mapping based on the EC 1.3.8.8 catalytic activity. ACAD9
      does have demonstrated long-chain acyl-CoA dehydrogenase activity (PMID:16020546), making
      this a valid annotation.
    action: ACCEPT
    reason: >-
      ACAD9 has experimentally demonstrated long-chain fatty acyl-CoA dehydrogenase activity
      with substrate preference for C16-C22 chain lengths (PMID:16020546). The IEA annotation
      correctly captures this function and is consistent with the IDA annotation from BHF-UCL.
    supported_by:
      - reference_id: PMID:16020546
        supporting_text: "Purified mature ACAD-9 had maximal activity with long-chain unsaturated acyl-CoAs as substrates (C16:1-, C18:1-, C18:2-, C22:6-CoA)"

# --- Annotation 4: mitochondrial inner membrane (IEA, SubCell) ---
- term:
    id: GO:0005743
    label: mitochondrial inner membrane
  evidence_type: IEA
  original_reference_id: GO_REF:0000044
  review:
    summary: >-
      IEA annotation based on UniProtKB/Swiss-Prot subcellular location mapping. ACAD9 is
      experimentally localized to the mitochondrial inner membrane, matrix side, as a peripheral
      membrane protein (PMID:16020546, PMID:20816094).
    action: ACCEPT
    reason: >-
      Multiple experimental studies confirm ACAD9 localizes to the mitochondrial inner membrane.
      Submitochondrial fractionation showed ACAD9 is membrane-associated (PMID:16020546). The
      UniProt entry explicitly states "Mitochondrion inner membrane; Peripheral membrane protein;
      Matrix side" with evidence from PMID:16020546 and PMID:20816094.
    supported_by:
      - reference_id: PMID:16020546
        supporting_text: "Submitochondrial fractionation studies found native ACAD-9 to be associated with the mitochondrial membrane"

# --- Annotation 5: fatty acid metabolic process (IEA, ARBA) ---
- term:
    id: GO:0006631
    label: fatty acid metabolic process
  evidence_type: IEA
  original_reference_id: GO_REF:0000117
  review:
    summary: >-
      IEA annotation from ARBA. ACAD9 participates in fatty acid beta-oxidation by catalyzing
      the first step of the FAO cycle. This is a broad term and while accurate, more specific
      terms (long-chain fatty acid metabolic process, GO:0001676) are already annotated.
    action: ACCEPT
    reason: >-
      ACAD9 does participate in fatty acid metabolism through its acyl-CoA dehydrogenase activity.
      While this IEA term is broader than the IDA annotation to GO:0001676, it is not incorrect
      and is an expected consequence of the ARBA mapping.
    supported_by:
      - reference_id: PMID:16020546
        supporting_text: "Human acyl-CoA dehydrogenase-9 plays a novel role in the mitochondrial beta-oxidation of unsaturated fatty acids"

# --- Annotation 6: oxidoreductase activity (IEA, KW) ---
- term:
    id: GO:0016491
    label: oxidoreductase activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000043
  review:
    summary: >-
      IEA annotation based on UniProtKB keyword mapping (KW-0560, Oxidoreductase). ACAD9 is
      indeed an oxidoreductase. This is a very general term but is not incorrect for an IEA.
    action: ACCEPT
    reason: >-
      ACAD9 is an oxidoreductase (acyl-CoA dehydrogenase, EC 1.3.8.7/1.3.8.8). While this
      is a very broad parent term, it is correctly applied via keyword mapping and more specific
      child terms are also annotated.
    supported_by:
      - reference_id: PMID:16020546
        supporting_text: "Purified mature ACAD-9 had maximal activity with long-chain unsaturated acyl-CoAs as substrates"

# --- Annotation 7: oxidoreductase activity, acting on the CH-CH group of donors (IEA, InterPro) ---
- term:
    id: GO:0016627
    label: oxidoreductase activity, acting on the CH-CH group of donors
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  review:
    summary: >-
      IEA annotation based on InterPro domain mapping. ACAD9 catalyzes alpha,beta-dehydrogenation
      of fatty acyl-CoA, which is an oxidoreductase reaction acting on CH-CH groups. This is a
      correct intermediate-level term between general oxidoreductase and specific acyl-CoA
      dehydrogenase activity.
    action: ACCEPT
    reason: >-
      The acyl-CoA dehydrogenase reaction catalyzed by ACAD9 involves oxidation of the CH-CH
      bond at the alpha-beta position of fatty acyl-CoA substrates. This InterPro-derived term
      is accurate and at an appropriate level of specificity for automated annotation.
    supported_by:
      - reference_id: PMID:16020546
        supporting_text: "Purified mature ACAD-9 had maximal activity with long-chain unsaturated acyl-CoAs as substrates"

# --- Annotation 8: mitochondrial membrane (IEA, ARBA) ---
- term:
    id: GO:0031966
    label: mitochondrial membrane
  evidence_type: IEA
  original_reference_id: GO_REF:0000117
  review:
    summary: >-
      IEA annotation from ARBA. ACAD9 is localized to the mitochondrial inner membrane. This
      term is broader (parent) than the more specific mitochondrial inner membrane annotations
      also present, but is not incorrect.
    action: ACCEPT
    reason: >-
      ACAD9 is experimentally localized to the mitochondrial membrane (specifically the inner
      membrane). This broader IEA term is subsumed by more specific annotations but remains valid.
    supported_by:
      - reference_id: PMID:16020546
        supporting_text: "Submitochondrial fractionation studies found native ACAD-9 to be associated with the mitochondrial membrane"

# --- Annotation 9: flavin adenine dinucleotide binding (IEA, InterPro) ---
- term:
    id: GO:0050660
    label: flavin adenine dinucleotide binding
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  review:
    summary: >-
      IEA annotation based on InterPro domain mapping. ACAD9 is a FAD-containing flavoprotein.
      FAD binding has been experimentally demonstrated, with approximately 70% FAD occupancy in
      purified protein (PMID:34646991). FAD binding is essential for the dehydrogenase function,
      and ECSIT binding causes deflavination (PMID:33320993, PMID:38086790).
    action: ACCEPT
    reason: >-
      ACAD9 binds FAD as a cofactor for its acyl-CoA dehydrogenase activity. The FAD content of
      purified ACAD9 is approximately 70% (PMID:34646991), and ECSIT-mediated deflavination
      switches ACAD9 from FAO enzyme to CI assembly factor (PMID:33320993).
    supported_by:
      - reference_id: PMID:34646991
        supporting_text: "the FAD content of purified wild type ACAD9 protein (non-tagged ACAD9 and ACAD9-His6) was approximately 70%"
      - reference_id: PMID:33320993
        supporting_text: "interaction with ECSIT induces ACAD9 to eject its FAD cofactor from the catalytic site"

# --- Annotation 10: medium-chain fatty acyl-CoA dehydrogenase activity (IEA, ARBA) ---
- term:
    id: GO:0070991
    label: medium-chain fatty acyl-CoA dehydrogenase activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  review:
    summary: >-
      IEA annotation from ARBA/Rhea mapping based on EC 1.3.8.7. ACAD9 does have some activity
      toward medium-chain substrates (C9-C11-CoA) as demonstrated in PMID:16020546. However,
      its primary specificity is for long-chain substrates; medium-chain activity is lower and
      secondary.
    action: KEEP_AS_NON_CORE
    reason: >-
      While ACAD9 is primarily a long-chain ACAD, it does have experimentally demonstrated
      medium-chain activity (PMID:16020546), and this is consistent with the EC 1.3.8.7
      assignment in UniProt. The IEA annotation correctly reflects the catalogued EC number.
    supported_by:
      - reference_id: PMID:16020546
        supporting_text: "Purified mature ACAD-9 had maximal activity with long-chain unsaturated acyl-CoAs as substrates (C16:1-, C18:1-, C18:2-, C22:6-CoA)"

# --- Annotation 11: very-long-chain fatty acyl-CoA dehydrogenase activity (IDA, PMID:33320993) ---
- term:
    id: GO:0017099
    label: very-long-chain fatty acyl-CoA dehydrogenase activity
  evidence_type: IDA
  original_reference_id: PMID:33320993
  review:
    summary: >-
      IDA annotation from FlyBase based on Giachin et al. 2021, which characterized ACAD9 acyl-CoA
      dehydrogenase activity using an ETF fluorescence reduction assay with palmitoyl-CoA (C16:0)
      as substrate. While ACAD9 does have dehydrogenase activity, the term "very-long-chain"
      (GO:0017099, typically C20+) may be an over-annotation. ACAD9's primary substrates are
      long-chain (C14-C18), not very-long-chain. The activity measured in this paper used
      palmitoyl-CoA (C16:0), which is a long-chain substrate.
    action: MODIFY
    reason: >-
      ACAD9 has optimal activity with long-chain (C14-C18) substrates, not very-long-chain (C20+).
      The paper PMID:33320993 demonstrated ACAD dehydrogenase activity using palmitoyl-CoA (C16:0),
      which is a long-chain substrate. While ACAD9 does have some activity on C20:0 (eicosanoyl-CoA)
      as shown in PMID:16020546, its primary specificity is long-chain. The annotation should be
      modified to long-chain fatty acyl-CoA dehydrogenase activity (GO:0004466).
    proposed_replacement_terms:
      - id: GO:0004466
        label: long-chain fatty acyl-CoA dehydrogenase activity
    supported_by:
      - reference_id: PMID:33320993
        supporting_text: "After addition of the ACAD specific substrate palmitoyl-CoA (C16:0), there is a clear loss of ETF fluorescence in ACAD9 alone"
      - reference_id: PMID:16020546
        supporting_text: "Purified mature ACAD-9 had maximal activity with long-chain unsaturated acyl-CoAs as substrates (C16:1-, C18:1-, C18:2-, C22:6-CoA)"

# --- Annotation 12: very-long-chain fatty acyl-CoA dehydrogenase activity (IDA, PMID:34646991) ---
- term:
    id: GO:0017099
    label: very-long-chain fatty acyl-CoA dehydrogenase activity
  evidence_type: IDA
  original_reference_id: PMID:34646991
  review:
    summary: >-
      IDA annotation from FlyBase based on Xia et al. 2021 (iScience). This paper characterized
      ACAD9 enzymatic activity using palmitoyl-CoA (C16:0) as substrate and measured dehydrogenation
      activity of 83 min-1. The activity is real but the term "very-long-chain" is an overstatement
      of specificity, since palmitoyl-CoA is a long-chain substrate.
    action: MODIFY
    reason: >-
      Same reasoning as above. ACAD9 has its optimal activity on long-chain substrates (C14-C18).
      The paper PMID:34646991 used palmitoyl-CoA (C16:0) as substrate, which is long-chain.
      The VLCAD-like annotation is misleading; ACAD9 activity is only 18% of VLCAD activity
      (PMID:34646991).
    proposed_replacement_terms:
      - id: GO:0004466
        label: long-chain fatty acyl-CoA dehydrogenase activity
    supported_by:
      - reference_id: PMID:34646991
        supporting_text: "this activity is only 18% of VLCAD activity"
      - reference_id: PMID:34646991
        supporting_text: "As purified, the wild-type ACAD9 protein has a dehydrogenation activity of 83 min-1, attributable to the FAD-containing holo-form of the protein"

# --- Annotation 13: protein-macromolecule adaptor activity (IDA, PMID:32320651) ---
- term:
    id: GO:0030674
    label: protein-macromolecule adaptor activity
  evidence_type: IDA
  original_reference_id: PMID:32320651
  review:
    summary: >-
      IDA annotation from FlyBase based on Formosa et al. 2020, which demonstrated that ACAD9
      is a core component of the MCIA complex and acts as an adaptor linking ECSIT (and through it,
      NDUFAF1) to CI assembly intermediates. The study showed ACAD9 forms a hierarchy of stability
      centered on itself within the MCIA complex. This is an excellent annotation capturing ACAD9's
      primary assembly factor role using a molecular function term.
    action: ACCEPT
    reason: >-
      ACAD9 functions as a protein-macromolecule adaptor within the MCIA complex, bridging ECSIT
      to CI assembly intermediates. This is well-supported by PMID:32320651, which showed that
      knockout of ACAD9 destabilizes the entire MCIA complex, and by structural studies showing
      ACAD9 provides binding surfaces for ECSIT via its vestigial dehydrogenase domain
      (PMID:33320993, PMID:38086790). This annotation captures the core molecular function of
      ACAD9 in CI assembly.
    supported_by:
      - reference_id: PMID:32320651
        supporting_text: "while each MCIA component is critical for complex I assembly, a hierarchy of stability exists centered on ACAD9"
      - reference_id: PMID:33320993
        supporting_text: "ECSIT functions as the bridging node of the MCIA core complex"

# --- Annotation 14: mitochondrial complex I intermediate assembly complex (IDA, PMID:33320993) ---
- term:
    id: GO:0160295
    label: mitochondrial complex I intermediate assembly complex
  evidence_type: IDA
  original_reference_id: PMID:33320993
  review:
    summary: >-
      IDA annotation from FlyBase based on Giachin et al. 2021. This study provided extensive
      structural and biochemical evidence that ACAD9 is a core component of the MCIA complex,
      including cryo-EM structures, SAXS, native MS, and SEC-MALLS data demonstrating stable
      ACAD9-ECSIT complexes.
    action: ACCEPT
    reason: >-
      ACAD9 is unequivocally a component of the MCIA complex. Giachin et al. demonstrated
      formation of the ACAD9-ECSIT binary complex by multiple biophysical methods and showed
      ECSIT binds the vestigial dehydrogenase domain of ACAD9 (PMID:33320993). This is a core
      annotation for ACAD9.
    supported_by:
      - reference_id: PMID:33320993
        supporting_text: "ECSIT functions as the bridging node of the MCIA core complex"
      - reference_id: PMID:33320993
        supporting_text: "the C-terminal domain of ECSIT directly binds to the vestigial dehydrogenase domain of the FAO enzyme ACAD9 and induces its deflavination"

# --- Annotation 15: mitochondrial complex I intermediate assembly complex (IDA, PMID:34646991) ---
- term:
    id: GO:0160295
    label: mitochondrial complex I intermediate assembly complex
  evidence_type: IDA
  original_reference_id: PMID:34646991
  review:
    summary: >-
      IDA annotation from FlyBase based on Xia et al. 2021 (iScience). This study reconstituted
      the ACAD9/ECSIT/NDUFAF1 ternary complex in vitro and characterized it by SEC, SAXS, and
      pull-down assays, demonstrating ACAD9 is a core member of the MCIA complex.
    action: ACCEPT
    reason: >-
      Xia et al. provided direct biochemical evidence for ACAD9 as part of the MCIA ternary
      complex with ECSIT and NDUFAF1, including purification of the stable ternary complex with
      1:1:1 stoichiometry (PMID:34646991). This is strong supporting evidence for the CC annotation.
    supported_by:
      - reference_id: PMID:34646991
        supporting_text: "ACAD9, ECSIT, and NDUFAF1 form the core mitochondrial CI assembly (MCIA) complex"
      - reference_id: PMID:34646991
        supporting_text: "mixing of the three purified, individual proteins forms a ternary complex, ACAD9/ECSIT/NDUFAF1, with a 1:1:1 molar (monomer) ratio"

# --- Annotation 16: mitochondrion (IDA, GO_REF:0000052, HPA) ---
- term:
    id: GO:0005739
    label: mitochondrion
  evidence_type: IDA
  original_reference_id: GO_REF:0000052
  review:
    summary: >-
      IDA annotation from HPA based on immunofluorescence data. ACAD9 localization to
      mitochondria is well-established across multiple studies and methods.
    action: ACCEPT
    reason: >-
      ACAD9 mitochondrial localization is confirmed by multiple independent approaches:
      submitochondrial fractionation (PMID:16020546), immunofluorescence (PMID:20816094),
      and proteomics (PMID:34800366). The HPA immunofluorescence data is consistent with
      this body of evidence.
    supported_by:
      - reference_id: PMID:16020546
        supporting_text: "Submitochondrial fractionation studies found native ACAD-9 to be associated with the mitochondrial membrane"

# --- Annotation 17: mitochondrial complex I intermediate assembly complex (IDA, PMID:32320651) ---
- term:
    id: GO:0160295
    label: mitochondrial complex I intermediate assembly complex
  evidence_type: IDA
  original_reference_id: PMID:32320651
  review:
    summary: >-
      IDA annotation from FlyBase based on Formosa et al. 2020. This study used cell knockout
      studies of each MCIA component and demonstrated that ACAD9 is central to MCIA complex
      stability, also identifying TMEM186 and COA1 as additional MCIA components.
    action: ACCEPT
    reason: >-
      Formosa et al. provided strong genetic evidence from knockout studies that ACAD9 is a
      core component of the MCIA complex, with a central role in maintaining complex stability.
      Loss of ACAD9 resulted in destabilization of the entire MCIA complex and impaired CI
      assembly (PMID:32320651).
    supported_by:
      - reference_id: PMID:32320651
        supporting_text: "The mitochondrial complex I intermediate assembly (MCIA) complex, containing assembly factors NDUFAF1, ECSIT, ACAD9, and TMEM126B, is required for building the intermediate ND2-module"
      - reference_id: PMID:32320651
        supporting_text: "while each MCIA component is critical for complex I assembly, a hierarchy of stability exists centered on ACAD9"

# --- Annotation 18: acyl-CoA dehydrogenase activity (IDA, PMID:38086790) ---
- term:
    id: GO:0003995
    label: acyl-CoA dehydrogenase activity
  evidence_type: IDA
  original_reference_id: PMID:38086790
  review:
    summary: >-
      IDA annotation from FlyBase based on McGregor et al. 2023 (Nature Communications). This
      study determined a 3.0 A cryo-EM structure of the ACAD9-ECSIT complex and also characterized
      ACAD9 dehydrogenase activity using the ETF fluorescence reduction assay with palmitoyl-CoA.
      The study demonstrated ECSIT binding induces a large conformational change in the FAD-binding
      loop of ACAD9, releasing FAD and converting ACAD9 from an FAO enzyme to a CI assembly factor.
    action: ACCEPT
    reason: >-
      McGregor et al. directly measured ACAD9 acyl-CoA dehydrogenase activity via ETF fluorescence
      reduction assay and confirmed the deflavination mechanism (PMID:38086790). This provides
      direct experimental evidence for ACAD9's enzymatic function.
    supported_by:
      - reference_id: PMID:38086790
        supporting_text: "ECSIT binding induces a major conformational change in the FAD-binding loop of ACAD9, releasing the FAD cofactor and converting ACAD9 from a fatty acid beta-oxidation (FAO) enzyme to a CI assembly factor"

# --- Annotation 19: mitochondrial inner membrane (NAS, PMID:32320651) ---
- term:
    id: GO:0005743
    label: mitochondrial inner membrane
  evidence_type: NAS
  original_reference_id: PMID:32320651
  review:
    summary: >-
      NAS annotation from ComplexPortal based on PMID:32320651. ACAD9 functions as part of the
      MCIA complex at the mitochondrial inner membrane where CI assembly occurs. This is
      consistent with the experimentally determined localization.
    action: ACCEPT
    reason: >-
      ACAD9 is localized to the mitochondrial inner membrane, matrix side, as a peripheral
      membrane protein. This is well-supported by fractionation studies (PMID:16020546) and
      is the site of its CI assembly function as part of the MCIA complex (PMID:32320651).
    supported_by:
      - reference_id: PMID:16020546
        supporting_text: "Submitochondrial fractionation studies found native ACAD-9 to be associated with the mitochondrial membrane"

# --- Annotation 20: mitochondrial respiratory chain complex I assembly (NAS, PMID:32320651) ---
- term:
    id: GO:0032981
    label: mitochondrial respiratory chain complex I assembly
  evidence_type: NAS
  original_reference_id: PMID:32320651
  review:
    summary: >-
      NAS annotation from ComplexPortal based on Formosa et al. 2020. ACAD9 is essential for
      CI assembly as a core member of the MCIA complex. This is the primary biological process
      annotation for ACAD9 and represents its most important physiological function.
    action: ACCEPT
    reason: >-
      CI assembly is the primary biological process function of ACAD9. Multiple studies demonstrate
      that loss of ACAD9 causes isolated CI deficiency (PMID:20816094), ACAD9 is part of the
      MCIA complex required for ND2-module assembly (PMID:32320651), and disease mutations
      cluster in the assembly domain (PMID:20816094).
    additional_reference_ids:
      - file:human/ACAD9/ACAD9-deep-research-falcon.md
    supported_by:
      - reference_id: PMID:32320651
        supporting_text: "The mitochondrial complex I intermediate assembly (MCIA) complex, containing assembly factors NDUFAF1, ECSIT, ACAD9, and TMEM126B, is required for building the intermediate ND2-module"
      - reference_id: PMID:20816094
        supporting_text: "ACAD9 binds complex I assembly factors NDUFAF1 and Ecsit and is specifically required for the assembly of complex I"
      - reference_id: file:human/ACAD9/ACAD9-deep-research-falcon.md
        supporting_text: "its dominant essential role is as a CI assembly factor within MCIA."

# --- Annotation 21: mitochondrion (HTP, PMID:34800366) ---
- term:
    id: GO:0005739
    label: mitochondrion
  evidence_type: HTP
  original_reference_id: PMID:34800366
  review:
    summary: >-
      HTP annotation from high-throughput human mitochondrial proteome study (Morgenstern et al.
      2021). ACAD9 was identified as a high-confidence mitochondrial protein. This is consistent
      with extensive prior evidence.
    action: ACCEPT
    reason: >-
      ACAD9 is a well-established mitochondrial protein. Its identification in a quantitative
      high-confidence mitochondrial proteome study (PMID:34800366) adds further confirmation
      to established knowledge from targeted studies (PMID:16020546, PMID:20816094).
    supported_by:
      - reference_id: PMID:16020546
        supporting_text: "Submitochondrial fractionation studies found native ACAD-9 to be associated with the mitochondrial membrane"

# --- Annotation 22: protein binding (IPI, PMID:33753518) ---
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:33753518
  review:
    summary: >-
      IPI annotation based on Carroll et al. 2021, which showed TMEM70 and TMEM242 interact
      with MCIA complex components including ACAD9. The WITH field includes UniProtKB:Q8IUX1
      (TMEM242), Q9BQ95 (TMEM126B), Q9BUB7 (TMEM70), and Q9Y375 (NDUFAF1). While the
      interactions are real and informative, the term 'protein binding' is uninformative.
      The adaptor activity annotation (GO:0030674) already captures the functionally relevant
      aspect.
    action: MARK_AS_OVER_ANNOTATED
    reason: >-
      The protein binding term is uninformative. The ACAD9 interactions with TMEM70 and TMEM242
      are real (PMID:33753518), but this function is better captured by the protein-macromolecule
      adaptor activity annotation (GO:0030674) and the MCIA complex membership (GO:0160295).
      Generic 'protein binding' does not tell us anything about the actual function of ACAD9.
    supported_by:
      - reference_id: PMID:33753518
        supporting_text: "TMEM70 and TMEM242 interact with the mitochondrial complex I assembly (the MCIA) complex that supports assembly of the membrane arm of complex I"

# --- Annotation 23: protein binding (IPI, PMID:32320651) ---
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:32320651
  review:
    summary: >-
      IPI annotation based on Formosa et al. 2020. The WITH field includes TMEM126B (Q96B77),
      ECSIT (Q9BQ95), TMEM186 (Q9GZY4), and NDUFAF1 (Q9Y375). These are the MCIA complex
      interactions. While real, 'protein binding' is uninformative.
    action: MARK_AS_OVER_ANNOTATED
    reason: >-
      Same as above. The interactions with MCIA complex components are real and well-characterized
      (PMID:32320651), but 'protein binding' is uninformative. The adaptor activity (GO:0030674)
      and MCIA complex membership (GO:0160295) annotations are more informative.
    supported_by:
      - reference_id: PMID:32320651
        supporting_text: "The mitochondrial complex I intermediate assembly (MCIA) complex, containing assembly factors NDUFAF1, ECSIT, ACAD9, and TMEM126B, is required for building the intermediate ND2-module"

# --- Annotation 24: long-chain fatty acid metabolic process (IDA, PMID:16020546) ---
- term:
    id: GO:0001676
    label: long-chain fatty acid metabolic process
  evidence_type: IDA
  original_reference_id: PMID:16020546
  review:
    summary: >-
      IDA annotation from BHF-UCL based on Ensenauer et al. 2005. ACAD9 participates in
      long-chain fatty acid beta-oxidation, with maximal activity toward long-chain unsaturated
      acyl-CoA substrates (C16:1, C18:1, C18:2, C22:6). This is a secondary/moonlighting
      function but is experimentally well-supported.
    action: KEEP_AS_NON_CORE
    reason: >-
      While ACAD9 does participate in long-chain fatty acid metabolism, this represents a
      secondary/moonlighting function. The primary in vivo role is CI assembly (PMID:20816094).
      Disease mutations cause CI deficiency rather than primary FAO defects (PMID:20816094).
      However, FAO activity is tissue-relevant especially in brain where ACAD9 is the main
      long-chain ACAD (PMID:16020546, PMID:17564966).
    supported_by:
      - reference_id: PMID:16020546
        supporting_text: "Purified mature ACAD-9 had maximal activity with long-chain unsaturated acyl-CoAs as substrates (C16:1-, C18:1-, C18:2-, C22:6-CoA)"
      - reference_id: PMID:20816094
        supporting_text: "ACAD9 mutations result in complex I deficiency and not in disturbed long-chain fatty acid oxidation"

# --- Annotation 25: long-chain fatty acyl-CoA dehydrogenase activity (IDA, PMID:16020546) ---
- term:
    id: GO:0004466
    label: long-chain fatty acyl-CoA dehydrogenase activity
  evidence_type: IDA
  original_reference_id: PMID:16020546
  review:
    summary: >-
      IDA annotation from BHF-UCL based on Ensenauer et al. 2005. ACAD9 was purified, expressed,
      and its substrate utilization pattern characterized, demonstrating maximal activity with
      long-chain (C16-C18) unsaturated acyl-CoA substrates. This is the most appropriate
      specific MF term for ACAD9's enzymatic activity.
    action: ACCEPT
    reason: >-
      ACAD9 has well-characterized long-chain acyl-CoA dehydrogenase activity with Km values
      of 2.8 uM for hexadecanoyl-CoA and 0.7 uM for (9Z)-hexadecenoyl-CoA (PMID:16020546).
      This is the correct level of specificity for ACAD9's enzymatic function.
    supported_by:
      - reference_id: PMID:16020546
        supporting_text: "Purified mature ACAD-9 had maximal activity with long-chain unsaturated acyl-CoAs as substrates (C16:1-, C18:1-, C18:2-, C22:6-CoA)"

# --- Annotation 26: mitochondrion (IDA, PMID:16020546) ---
- term:
    id: GO:0005739
    label: mitochondrion
  evidence_type: IDA
  original_reference_id: PMID:16020546
  review:
    summary: >-
      IDA annotation from BHF-UCL based on Ensenauer et al. 2005. Submitochondrial fractionation
      studies confirmed ACAD9 is associated with the mitochondrial membrane.
    action: ACCEPT
    reason: >-
      Direct experimental evidence from submitochondrial fractionation demonstrates ACAD9
      is a mitochondrial protein associated with mitochondrial membranes (PMID:16020546).
    supported_by:
      - reference_id: PMID:16020546
        supporting_text: "Submitochondrial fractionation studies found native ACAD-9 to be associated with the mitochondrial membrane"

# --- Annotation 27: mitochondrial membrane (IDA, PMID:16020546) ---
- term:
    id: GO:0031966
    label: mitochondrial membrane
  evidence_type: IDA
  original_reference_id: PMID:16020546
  review:
    summary: >-
      IDA annotation from BHF-UCL based on Ensenauer et al. 2005. Submitochondrial fractionation
      showed ACAD9 is membrane-associated. This is less specific than the inner membrane annotation
      but still correct based on the fractionation data.
    action: ACCEPT
    reason: >-
      ACAD9 is a peripheral membrane protein associated with the mitochondrial inner membrane
      on the matrix side (PMID:16020546). The mitochondrial membrane annotation is correct,
      though the more specific inner membrane annotation is also present.
    supported_by:
      - reference_id: PMID:16020546
        supporting_text: "Submitochondrial fractionation studies found native ACAD-9 to be associated with the mitochondrial membrane"

# --- Annotation 28: medium-chain fatty acid metabolic process (IDA, PMID:16020546) ---
- term:
    id: GO:0051791
    label: medium-chain fatty acid metabolic process
  evidence_type: IDA
  original_reference_id: PMID:16020546
  review:
    summary: >-
      IDA annotation from BHF-UCL based on Ensenauer et al. 2005. ACAD9 shows some activity
      toward medium-chain substrates (C9-C11-CoA) in addition to its primary long-chain
      specificity. This is a secondary activity.
    action: KEEP_AS_NON_CORE
    reason: >-
      ACAD9 does have some medium-chain acyl-CoA dehydrogenase activity (PMID:16020546), but
      its primary enzymatic specificity is for long-chain substrates, and its primary biological
      role is CI assembly. Medium-chain FAO is a minor secondary activity, better handled by
      MCAD in vivo.
    supported_by:
      - reference_id: PMID:16020546
        supporting_text: "Purified mature ACAD-9 had maximal activity with long-chain unsaturated acyl-CoAs as substrates (C16:1-, C18:1-, C18:2-, C22:6-CoA)"

# --- Annotation 29: medium-chain fatty acyl-CoA dehydrogenase activity (IDA, PMID:16020546) ---
- term:
    id: GO:0070991
    label: medium-chain fatty acyl-CoA dehydrogenase activity
  evidence_type: IDA
  original_reference_id: PMID:16020546
  review:
    summary: >-
      IDA annotation from BHF-UCL based on Ensenauer et al. 2005. ACAD9 shows activity toward
      some medium-chain substrates in the C9-C11 range. While less active than on long-chain
      substrates, this has been directly demonstrated experimentally.
    action: KEEP_AS_NON_CORE
    reason: >-
      ACAD9 does have demonstrable medium-chain acyl-CoA dehydrogenase activity (PMID:16020546),
      though this is secondary to its long-chain specificity and far secondary to its CI assembly
      role. The activity is real but minor compared to dedicated MCAD.
    supported_by:
      - reference_id: PMID:16020546
        supporting_text: "Purified mature ACAD-9 had maximal activity with long-chain unsaturated acyl-CoAs as substrates (C16:1-, C18:1-, C18:2-, C22:6-CoA)"

# --- Annotation 30: mitochondrial inner membrane (TAS, Reactome:R-HSA-6799179) ---
- term:
    id: GO:0005743
    label: mitochondrial inner membrane
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-6799179
  review:
    summary: >-
      TAS annotation from Reactome pathway "Peripheral arm subunits bind the 815kDa complex to
      form a 980kDa complex". ACAD9 functions at the mitochondrial inner membrane as part of the
      MCIA complex during CI biogenesis.
    action: ACCEPT
    reason: >-
      ACAD9 is localized to the mitochondrial inner membrane where CI assembly takes place.
      This is well-supported experimentally (PMID:16020546, PMID:20816094) and the Reactome
      annotation correctly reflects this.
    supported_by:
      - reference_id: PMID:16020546
        supporting_text: "Submitochondrial fractionation studies found native ACAD-9 to be associated with the mitochondrial membrane"

# --- Annotation 31: mitochondrial inner membrane (TAS, Reactome:R-HSA-6799196) ---
- term:
    id: GO:0005743
    label: mitochondrial inner membrane
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-6799196
  review:
    summary: >-
      TAS annotation from Reactome pathway "The MCIA complex, NDUFAF2-7 all dissociate from the
      980kDa complex, resulting in Complex I". Consistent with ACAD9's role as an MCIA assembly
      factor at the inner membrane.
    action: ACCEPT
    reason: >-
      Same as above. ACAD9 is at the mitochondrial inner membrane and this Reactome step
      describes MCIA complex dissociation after CI assembly is complete. Well-supported
      experimentally.
    supported_by:
      - reference_id: PMID:16020546
        supporting_text: "Submitochondrial fractionation studies found native ACAD-9 to be associated with the mitochondrial membrane"

# --- Annotation 32: mitochondrial inner membrane (TAS, Reactome:R-HSA-6799197) ---
- term:
    id: GO:0005743
    label: mitochondrial inner membrane
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-6799197
  review:
    summary: >-
      TAS annotation from Reactome pathway "ND4, ND5 bind the 550kDa complex to form the 815kDa
      complex". Another step in CI biogenesis at the inner membrane.
    action: ACCEPT
    reason: >-
      Consistent with ACAD9's role as an MCIA assembly factor at the mitochondrial inner membrane.
    supported_by:
      - reference_id: PMID:16020546
        supporting_text: "Submitochondrial fractionation studies found native ACAD-9 to be associated with the mitochondrial membrane"

# --- Annotation 33: mitochondrial inner membrane (TAS, Reactome:R-HSA-6799199) ---
- term:
    id: GO:0005743
    label: mitochondrial inner membrane
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-6799199
  review:
    summary: >-
      TAS annotation from Reactome pathway "COA1:MT-ND2, TMEM186:MT-ND3, MT-ND6, NDUFB6 bind
      the MCIA complex to form a 370kDa subcomplex". This step directly involves the MCIA complex
      of which ACAD9 is a core member.
    action: ACCEPT
    reason: >-
      ACAD9 is a core member of the MCIA complex that participates in this assembly step.
      Well-supported by PMID:32320651.
    supported_by:
      - reference_id: PMID:32320651
        supporting_text: "The mitochondrial complex I intermediate assembly (MCIA) complex, containing assembly factors NDUFAF1, ECSIT, ACAD9, and TMEM126B, is required for building the intermediate ND2-module"

# --- Annotation 34: mitochondrial inner membrane (TAS, Reactome:R-HSA-6799202) ---
- term:
    id: GO:0005743
    label: mitochondrial inner membrane
  evidence_type: TAS
  original_reference_id: Reactome:R-HSA-6799202
  review:
    summary: >-
      TAS annotation from Reactome pathway "The 315kDa subcomplex binds the 370kDa subcomplex
      to form the 550kDa complex". Another CI assembly step at the inner membrane.
    action: ACCEPT
    reason: >-
      Consistent with ACAD9's role as an MCIA assembly factor at the mitochondrial inner membrane.
    supported_by:
      - reference_id: PMID:16020546
        supporting_text: "Submitochondrial fractionation studies found native ACAD-9 to be associated with the mitochondrial membrane"

# --- Annotation 35: protein binding (IPI, PMID:20816094) ---
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:20816094
  review:
    summary: >-
      IPI annotation based on Nouws et al. 2010, which showed ACAD9 interacts with NDUFAF1
      (Q9Y375) and ECSIT (Q9BQ95). These are the foundational interactions establishing ACAD9
      as an MCIA complex member. However, 'protein binding' is uninformative.
    action: MARK_AS_OVER_ANNOTATED
    reason: >-
      The interactions demonstrated by Nouws et al. (PMID:20816094) are central to understanding
      ACAD9's CI assembly function, but the generic 'protein binding' term fails to capture the
      biological significance. The adaptor activity (GO:0030674) and MCIA complex membership
      (GO:0160295) annotations are far more informative.
    supported_by:
      - reference_id: PMID:20816094
        supporting_text: "ACAD9 binds complex I assembly factors NDUFAF1 and Ecsit and is specifically required for the assembly of complex I"

# --- Annotation 36: mitochondrion (IDA, PMID:20816094) ---
- term:
    id: GO:0005739
    label: mitochondrion
  evidence_type: IDA
  original_reference_id: PMID:20816094
  review:
    summary: >-
      IDA annotation based on Nouws et al. 2010. ACAD9 was shown to localize to mitochondria
      as part of the study establishing its role in CI assembly.
    action: ACCEPT
    reason: >-
      ACAD9 mitochondrial localization is well-established and confirmed in this study
      (PMID:20816094) which demonstrated ACAD9 interactions with mitochondrial CI assembly
      factors.
    supported_by:
      - reference_id: PMID:20816094
        supporting_text: "ACAD9 binds complex I assembly factors NDUFAF1 and Ecsit and is specifically required for the assembly of complex I"

# --- Annotation 37: mitochondrial respiratory chain complex I assembly (IMP, PMID:20816094) ---
- term:
    id: GO:0032981
    label: mitochondrial respiratory chain complex I assembly
  evidence_type: IMP
  original_reference_id: PMID:20816094
  review:
    summary: >-
      IMP annotation based on Nouws et al. 2010. This landmark study demonstrated that ACAD9
      mutations result in CI deficiency (not FAO deficiency), establishing CI assembly as ACAD9's
      primary function. ACAD9 knockdown/mutation leads to impaired CI assembly. This is the
      strongest evidence for ACAD9's primary biological process.
    action: ACCEPT
    reason: >-
      Nouws et al. provided the foundational evidence that ACAD9 is required for CI biogenesis,
      showing that ACAD9 mutations cause CI deficiency rather than FAO defects, and that ACAD9
      interacts with known CI assembly factors NDUFAF1 and ECSIT (PMID:20816094). This IMP
      annotation is the most critical biological process annotation for ACAD9.
    supported_by:
      - reference_id: PMID:20816094
        supporting_text: "ACAD9 binds complex I assembly factors NDUFAF1 and Ecsit and is specifically required for the assembly of complex I"
      - reference_id: PMID:20816094
        supporting_text: "ACAD9 mutations result in complex I deficiency and not in disturbed long-chain fatty acid oxidation"

# --- Annotation 38: nucleus (IDA, PMID:21237683) ---
- term:
    id: GO:0005634
    label: nucleus
  evidence_type: IDA
  original_reference_id: PMID:21237683
  review:
    summary: >-
      IDA annotation based on He et al. 2011. This study characterized new long-chain ACADs
      including ACAD9, ACAD10, and ACAD11, and reported ACAD9 in the nucleus. However, ACAD9
      is a mitochondrial protein with a mitochondrial transit peptide, and all functional studies
      place it in mitochondria. Nuclear localization is likely an artifact of overexpression or
      immunostaining cross-reactivity and is not supported by the consensus of literature.
    action: REMOVE
    reason: >-
      ACAD9 has a mitochondrial transit peptide (residues 1-37, cleaved upon import; PMID:16020546)
      and all functional characterization places it in mitochondria. The nuclear localization
      reported in PMID:21237683 is not supported by other studies and is likely artifactual.
      UniProt does not include nuclear localization. The extensive proteomics and fractionation
      data consistently place ACAD9 in mitochondria only.
    supported_by:
      - reference_id: PMID:16020546
        supporting_text: "A 37-amino acid leader peptide was cleaved sequentially by two mitochondrial peptidases to yield a predicted molecular mass of 65 kDa for the mature subunit"

# --- Annotation 39: dendrite (IDA, PMID:21237683) ---
- term:
    id: GO:0030425
    label: dendrite
  evidence_type: IDA
  original_reference_id: PMID:21237683
  review:
    summary: >-
      IDA annotation based on He et al. 2011. The study examined ACAD expression patterns in
      human cerebellum and found ACAD9 was highly expressed in the granular layer. The dendrite
      annotation likely reflects immunohistochemical staining of neuronal processes. However,
      this is a cell-type expression pattern rather than a specific subcellular localization
      to dendrites. The protein within those cells is still in mitochondria.
    action: REMOVE
    reason: >-
      ACAD9 is a mitochondrial matrix-facing protein. Finding it in neurons (including dendritic
      processes) reflects tissue expression (ACAD9 is highly expressed in cerebellum) rather than
      specific dendritic localization. The protein is in mitochondria within dendrites, not in
      the dendritic cytoplasm. This annotation conflates cell-type expression with subcellular
      localization.
    supported_by:
      - reference_id: PMID:21237683
        supporting_text: "ACAD9 was most highly expressed in the granular layer"
      - reference_id: PMID:16020546
        supporting_text: "Submitochondrial fractionation studies found native ACAD-9 to be associated with the mitochondrial membrane"

# --- Annotation 40: mitochondrion (IDA, GO_REF:0000054, LIFEdb) ---
- term:
    id: GO:0005739
    label: mitochondrion
  evidence_type: IDA
  original_reference_id: GO_REF:0000054
  review:
    summary: >-
      IDA annotation from LIFEdb based on expressed fusion protein localization in living cells.
      ACAD9 localization to mitochondria was confirmed, consistent with all other evidence.
    action: ACCEPT
    reason: >-
      ACAD9 mitochondrial localization is well-established. The LIFEdb fusion protein experiment
      confirms what is known from multiple other approaches (PMID:16020546, PMID:20816094,
      PMID:34800366).
    supported_by:
      - reference_id: PMID:16020546
        supporting_text: "Submitochondrial fractionation studies found native ACAD-9 to be associated with the mitochondrial membrane"

# --- NEW annotations not currently in GOA ---
# --- Annotation 41: NEW - fatty acid beta-oxidation (BP) ---
- term:
    id: GO:0006635
    label: fatty acid beta-oxidation
  evidence_type: IDA
  original_reference_id: PMID:16020546
  review:
    summary: >-
      ACAD9 catalyzes the first step of mitochondrial fatty acid beta-oxidation, the
      alpha,beta-dehydrogenation of long-chain fatty acyl-CoA substrates. This specific
      biological process term is not currently annotated but is more precise than the existing
      GO:0006631 (fatty acid metabolic process) and GO:0001676 (long-chain fatty acid metabolic
      process) annotations.
    action: NEW
    reason: >-
      ACAD9 directly catalyzes the first step of the beta-oxidation cycle. The existing BP
      annotations (GO:0006631 fatty acid metabolic process, GO:0001676 long-chain fatty acid
      metabolic process) are correct but the more specific fatty acid beta-oxidation term is
      warranted given the direct experimental evidence. This is ACAD9's secondary core function.
    supported_by:
      - reference_id: PMID:16020546
        supporting_text: "Human acyl-CoA dehydrogenase-9 plays a novel role in the mitochondrial beta-oxidation of unsaturated fatty acids"
      - reference_id: PMID:34646991
        supporting_text: "ACAD9 was first identified as a member of the acyl-CoA dehydrogenase family catalyzing the alpha, beta-dehydrogenation of fatty acyl-CoA thioesters, the first step of the fatty acid beta-oxidation cycle"

core_functions:
- molecular_function:
    id: GO:0030674
    label: protein-macromolecule adaptor activity
  directly_involved_in:
  - id: GO:0032981
    label: mitochondrial respiratory chain complex I assembly
  locations:
  - id: GO:0005743
    label: mitochondrial inner membrane
  in_complex:
    id: GO:0160295
    label: mitochondrial complex I intermediate assembly complex
  description: >-
    Primary function. ACAD9 is a core component of the MCIA complex, functioning as a
    protein-macromolecule adaptor that bridges ECSIT (and through ECSIT, NDUFAF1) to CI assembly
    intermediates. ECSIT binding triggers deflavination and a conformational switch from FAO enzyme
    to assembly factor. Disease mutations causing CI deficiency (MC1DN20) underscore this as the
    essential role.
  supported_by:
  - reference_id: PMID:20816094
    supporting_text: "ACAD9 binds complex I assembly factors NDUFAF1 and Ecsit and is specifically required for the assembly of complex I"
  - reference_id: PMID:32320651
    supporting_text: "while each MCIA component is critical for complex I assembly, a hierarchy of stability exists centered on ACAD9"
  - reference_id: PMID:38086790
    supporting_text: "ECSIT binding induces a major conformational change in the FAD-binding loop of ACAD9, releasing the FAD cofactor and converting ACAD9 from a fatty acid beta-oxidation (FAO) enzyme to a CI assembly factor"

- molecular_function:
    id: GO:0004466
    label: long-chain fatty acyl-CoA dehydrogenase activity
  directly_involved_in:
  - id: GO:0006635
    label: fatty acid beta-oxidation
  locations:
  - id: GO:0005743
    label: mitochondrial inner membrane
  description: >-
    Secondary/moonlighting function. ACAD9 retains FAD-dependent acyl-CoA dehydrogenase activity
    (EC 1.3.8.8) toward long-chain fatty acyl-CoA substrates, catalyzing the first step of
    mitochondrial fatty acid beta-oxidation. Its activity is approximately 18% that of VLCAD. This
    function is mutually exclusive with the CI assembly role (ECSIT binding causes deflavination).
    ACAD9's FAO role is particularly relevant in tissues where it is the main long-chain ACAD, such
    as the CNS.
  supported_by:
  - reference_id: PMID:16020546
    supporting_text: "Purified mature ACAD-9 had maximal activity with long-chain unsaturated acyl-CoAs as substrates (C16:1-, C18:1-, C18:2-, C22:6-CoA)"
  - reference_id: PMID:34646991
    supporting_text: "this activity is only 18% of VLCAD activity"

proposed_new_terms: []

suggested_questions:
- question: >-
    What is the relative abundance of ACAD9 in its FAO (holo, FAD-bound) vs. CI assembly
    (apo, ECSIT-bound) states in different human tissues? Current understanding is based
    primarily on in vitro reconstitution, and the relative partitioning between FAO and
    assembly functions in vivo remains poorly quantified.

- question: >-
    Is there evidence that ACAD9 FAO function contributes to disease severity in ACAD9
    deficiency, beyond the CI assembly defect? Schiff et al. 2015 suggested both CI assembly
    and FAO enzyme activity contribute to disease severity, but the relative contribution
    remains debated.

suggested_experiments:
- description: >-
    Quantitative proteomics to measure the fraction of ACAD9 in ECSIT-bound (assembly) vs.
    free (FAO-competent) states across human tissues, especially brain regions. This would
    clarify the in vivo partitioning of ACAD9 between its two mutually exclusive functions.

references:
- id: GO_REF:0000002
  title: Gene Ontology annotation through association of InterPro records with GO terms
  findings: []
- id: GO_REF:0000033
  title: Annotation inferences using phylogenetic trees
  findings: []
- id: GO_REF:0000043
  title: Gene Ontology annotation based on UniProtKB/Swiss-Prot keyword mapping
  findings: []
- id: GO_REF:0000044
  title: >-
    Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary
    mapping, accompanied by conservative changes to GO terms applied by UniProt
  findings: []
- id: GO_REF:0000052
  title: Gene Ontology annotation based on curation of immunofluorescence data
  findings: []
- id: GO_REF:0000054
  title: >-
    Gene Ontology annotation based on curation of intracellular localizations of expressed
    fusion proteins in living cells
  findings: []
- id: GO_REF:0000117
  title: Electronic Gene Ontology annotations created by ARBA machine learning models
  findings: []
- id: GO_REF:0000120
  title: Combined Automated Annotation using Multiple IEA Methods
  findings: []
- id: PMID:16020546
  title: >-
    Human acyl-CoA dehydrogenase-9 plays a novel role in the mitochondrial beta-oxidation
    of unsaturated fatty acids.
  findings:
    - statement: >-
        ACAD9 is a mitochondrial membrane-associated homodimer with long-chain acyl-CoA
        dehydrogenase activity, maximal toward unsaturated C16-C22 substrates.
      supporting_text: >-
        Purified mature ACAD-9 had maximal activity with long-chain unsaturated acyl-CoAs
        as substrates (C16:1-, C18:1-, C18:2-, C22:6-CoA).
- id: PMID:20816094
  title: >-
    Acyl-CoA dehydrogenase 9 is required for the biogenesis of oxidative phosphorylation
    complex I.
  findings:
    - statement: >-
        ACAD9 binds CI assembly factors NDUFAF1 and ECSIT and is specifically required for CI
        assembly. ACAD9 mutations cause CI deficiency, not FAO defects.
      supporting_text: >-
        ACAD9 binds complex I assembly factors NDUFAF1 and Ecsit and is specifically required
        for the assembly of complex I. Furthermore, ACAD9 mutations result in complex I
        deficiency and not in disturbed long-chain fatty acid oxidation.
- id: PMID:21237683
  title: Identification and characterization of new long chain acyl-CoA dehydrogenases.
  findings:
    - statement: >-
        ACAD9 is highly expressed in human cerebellum (granular layer), where ACADV is not found,
        suggesting tissue-specific roles in long-chain FAO.
      supporting_text: >-
        ACAD9 was most highly expressed in the granular layer, ACAD11 in the white matter,
        and MCAD in the molecular layer and axons of specific neurons.
- id: PMID:24158852
  title: >-
    ACAD9, a complex I assembly factor with a moonlighting function in fatty acid oxidation
    deficiencies.
  findings:
    - statement: >-
        The CI assembly function of ACAD9 is independent of its FAO activity. The E426Q catalytic
        mutant abolishes dehydrogenase activity but does not affect CI assembly.
      supporting_text: >-
        Catalytically inactive ACAD9 gave partial-to-complete rescue of complex I
        biogenesis in ACAD9-deficient cells
- id: PMID:32320651
  title: >-
    Dissecting the Roles of Mitochondrial Complex I Intermediate Assembly Complex Factors in
    the Biogenesis of Complex I.
  findings:
    - statement: >-
        ACAD9 is a core member of the MCIA complex (with NDUFAF1, ECSIT, TMEM126B) required for
        ND2-module assembly. A hierarchy of stability exists centered on ACAD9. TMEM186 and COA1
        are additional MCIA components.
      supporting_text: >-
        while each MCIA component is critical for complex I assembly, a hierarchy of stability
        exists centered on ACAD9.
- id: PMID:33320993
  title: >-
    Assembly of The Mitochondrial Complex I Assembly Complex Suggests a Regulatory Role for
    Deflavination.
  findings:
    - statement: >-
        ECSIT C-terminal domain binds the vestigial dehydrogenase domain of ACAD9 and induces
        deflavination, switching ACAD9 from an FAO enzyme to a CI assembly factor. Cryo-EM
        confirmed the FAD binding site is empty in the ECSIT-bound state.
      supporting_text: >-
        interaction with ECSIT induces ACAD9 to eject its FAD cofactor from the catalytic site.
- id: PMID:33753518
  title: >-
    TMEM70 and TMEM242 help to assemble the rotor ring of human ATP synthase and interact
    with assembly factors for complex I.
  findings:
    - statement: >-
        TMEM70 and TMEM242 interact with MCIA complex components including ACAD9, ECSIT,
        NDUFAF1, and TMEM126B.
      supporting_text: >-
        When TMEM242-t and similarly tagged TMEM70 (TMEM70-t) were expressed separately in
        HEK293 cells, the most significantly associated proteins were ACAD9 (acyl-CoA
        dehydrogenase family member 9), ECSIT (evolutionarily conserved signaling intermediate
        in Toll pathway), and NDUFAF1 (NADH:ubiquinone oxidoreductase complex assembly factor 1)
- id: PMID:34646991
  title: >-
    Molecular mechanism of interactions between ACAD9 and binding partners in mitochondrial
    respiratory complex I assembly.
  findings:
    - statement: >-
        ACAD9, ECSIT, and NDUFAF1 form a stable 1:1:1 ternary complex. ECSIT binds at the ETF
        binding site in ACAD9's N-terminal domain, explaining the mutual exclusivity of FAO and
        assembly functions. ACAD9 activity is only 18% of VLCAD.
      supporting_text: >-
        this activity is only 18% of VLCAD activity
- id: PMID:34800366
  title: >-
    Quantitative high-confidence human mitochondrial proteome and its dynamics in cellular
    context.
  findings:
    - statement: >-
        ACAD9 was identified as a high-confidence member of the human mitochondrial proteome.
      supporting_text: >-
        We classified >8,000 proteins in mitochondrial preparations of human cells and
        defined a mitochondrial high-confidence proteome of >1,100 proteins (MitoCoP).
- id: PMID:38086790
  title: >-
    The assembly of the Mitochondrial Complex I Assembly complex uncovers a redox pathway
    coordination.
  findings:
    - statement: >-
        3.0 A cryo-EM structure of ACAD9-ECSIT complex revealed a 15-residue ECSIT peptide
        (res 320-334) that inserts at the junction of ACAD9 dehydrogenase and vestigial domains.
        ECSIT binding induces a 10 A gatekeeper loop movement in ACAD9, releasing FAD. ECSIT
        phosphorylation downregulates its association with ACAD9 and is reduced upon amyloid-beta
        exposure.
      supporting_text: >-
        ECSIT binding induces a major conformational change in the FAD-binding loop of ACAD9,
        releasing the FAD cofactor and converting ACAD9 from a fatty acid beta-oxidation (FAO)
        enzyme to a CI assembly factor.
- id: Reactome:R-HSA-6799179
  title: Peripheral arm subunits bind the 815kDa complex to form a 980kDa complex
  findings: []
- id: Reactome:R-HSA-6799196
  title: >-
    The MCIA complex, NDUFAF2-7 all dissociate from the 980kDa complex, resulting in Complex I
  findings: []
- id: Reactome:R-HSA-6799197
  title: ND4, ND5 bind the 550kDa complex to form the 815kDa complex
  findings: []
- id: Reactome:R-HSA-6799199
  title: >-
    COA1:MT-ND2, TMEM186:MT-ND3, MT-ND6, NDUFB6 bind the MCIA complex to form a 370kDa
    subcomplex
  findings: []
- id: Reactome:R-HSA-6799202
  title: The 315kDa subcomplex binds the 370kDa subcomplex to form the 550kDa complex
  findings: []