id: Q86TL0
gene_symbol: ATG4D
product_type: PROTEIN
status: COMPLETE
taxon:
  id: NCBITaxon:9606
  label: Homo sapiens
description: >-
  ATG4D is a cysteine protease of the peptidase C54 family that plays a key role in
  autophagy
  by mediating both proteolytic activation (priming) and delipidation of ATG8-family
  proteins
  (LC3/GABARAP). While ATG4D shows weak priming activity compared to ATG4B, it constitutes
  the major protein for delipidation activity in cells, particularly for LC3B. ATG4D
  can
  remove ATG8 from both PE and PS conjugates, functioning in both canonical macroautophagy
  and noncanonical single-membrane ATG8 conjugation (CASM). The protein contains a
  cryptic
  mitochondrial targeting sequence that is exposed upon caspase-3 cleavage at DEVD63,
  leading to mitochondrial import and roles in mitophagy/mitochondrial quality control.
  Human biallelic ATG4D variants are linked to neurodevelopmental disorders with
  cerebellar involvement.
existing_annotations:
  - term:
      id: GO:0000045
      label: autophagosome assembly
    evidence_type: IBA
    original_reference_id: GO_REF:0000033
    review:
      summary: >-
        ATG4D functions in autophagosome assembly through its role in processing ATG8-family
        proteins (LC3/GABARAP). The ATG4 family proteins drive phagophore growth,
        and ATG4D
        specifically promotes phagophore-ER contacts during the lipid transfer phase
        of
        autophagosome formation. IBA annotation is phylogenetically supported.
      action: ACCEPT
      reason: >-
        Core autophagy function well-supported by literature. ATG4D acts in autophagosome
        biogenesis through ATG8 processing and has additional protease-independent
        roles
        in phagophore growth via ATG9A trafficking regulation (PMID:33773106).
      supported_by:
        - reference_id: PMID:33773106
          supporting_text: >-
            ATG4 family proteins drive phagophore growth independently of the LC3/GABARAP
            lipidation system... ATG4s promote phagophore-ER contacts during the lipid-transfer
            phase of autophagosome formation
        - reference_id: file:human/ATG4D/ATG4D-deep-research-falcon.md
          supporting_text: 'model: Edison Scientific Literature'
  - term:
      id: GO:0004197
      label: cysteine-type endopeptidase activity
    evidence_type: IBA
    original_reference_id: GO_REF:0000033
    review:
      summary: >-
        ATG4D is a papain-like cysteine endopeptidase belonging to the peptidase C54
        family.
        It cleaves the C-terminal amino acids of ATG8 proteins (LC3/GABARAP) to expose
        the
        glycine required for lipidation. Kinetic studies demonstrate endopeptidase
        activity
        against MAP1LC3B (Km=13.1 uM) and GABARAPL2 (Km=7.2 uM).
      action: ACCEPT
      reason: >-
        Core molecular function directly demonstrated by biochemical studies. ATG4D
        contains
        the catalytic cysteine (Cys144) characteristic of C54 family proteases and
        has
        experimentally validated endopeptidase activity (PMID:21177865).
      supported_by:
        - reference_id: PMID:21177865
          supporting_text: >-
            The data indicated that Atg4B possessed the broadest spectrum against
            all substrates,
            followed by Atg4A, whereas Atg4C and Atg4D had minimal activities
  - term:
      id: GO:0005737
      label: cytoplasm
    evidence_type: IBA
    original_reference_id: GO_REF:0000033
    review:
      summary: >-
        ATG4D localizes to the cytoplasm where it performs its primary function of
        processing
        ATG8-family proteins on autophagosomal membranes. The full-length protein
        resides
        in cytoplasm where it functions in delipidation of autophagosomal ATG8.
      action: ACCEPT
      reason: >-
        Cytoplasmic localization is the primary site of ATG4D function. The deep research
        review confirms ATG4D acts on autophagosomal/autolysosomal membranes in the
        cytoplasm.
      additional_reference_ids:
        - file:human/ATG4D/ATG4D-deep-research-falcon.md
  - term:
      id: GO:0000423
      label: mitophagy
    evidence_type: IBA
    original_reference_id: GO_REF:0000033
    review:
      summary: >-
        ATG4D plays a role in mitophagy. ATG4D re-expression restores mitophagy more
        effectively
        than ATG4B in ATG4-quadruple knockout cells. The caspase-cleaved form (DeltaN63)
        targets
        mitochondria and associates with cardiolipin, supporting function in mitochondrial
        quality control.
      action: ACCEPT
      reason: >-
        Mitophagy function is well-supported by experimental evidence. ATG4D is involved
        in
        phagophore growth during mitophagy independently of its protease activity
        and of ATG8
        proteins by regulating ATG9A trafficking to mitochondria (PMID:33773106).
      supported_by:
        - reference_id: PMID:33773106
          supporting_text: >-
            ATG4 proximity networks reveal a role for ATG4s and their proximity partners,
            including the immune-disease protein LRBA, in ATG9A vesicle trafficking
            to mitochondria
  - term:
      id: GO:0016485
      label: protein processing
    evidence_type: IBA
    original_reference_id: GO_REF:0000033
    review:
      summary: >-
        ATG4D processes ATG8-family proteins by cleaving their C-terminus to expose
        the glycine
        required for lipidation (priming activity). This protein processing is essential
        for
        autophagy.
      action: ACCEPT
      reason: >-
        Core function of ATG4D family. While ATG4D has weak priming activity compared
        to ATG4B,
        it contributes to LC3/GABARAP processing in cells, especially GABARAPL1 and
        GABARAPL2
        (PMID:30661429).
      supported_by:
        - reference_id: PMID:30661429
          supporting_text: >-
            By further depletion of ATG4 isoforms, we discover that ATG4A, ATG4C and
            ATGD all
            contribute to the remaining processing activity and thus show overlapping
            redundancy
            in cells
  - term:
      id: GO:0019786
      label: protein-phosphatidylethanolamide deconjugating activity
    evidence_type: IBA
    original_reference_id: GO_REF:0000033
    review:
      summary: >-
        ATG4D catalyzes delipidation of PE-conjugated ATG8 proteins, removing them
        from membranes.
        This is a major in vivo function of ATG4D - it is the primary delipidating
        enzyme for
        LC3B in cells, more important than ATG4B for this function.
      action: ACCEPT
      reason: >-
        Core molecular function directly demonstrated. ATG4D constitutes the major
        protein for
        delipidation activity while having weak priming activity (PMID:29458288).
      supported_by:
        - reference_id: PMID:29458288
          supporting_text: >-
            all 4 ATG4 proteases can be activated to similar enzymatic activities
            on
            lipid-attached substrates... We suggest a model whereby ATG4B drives very
            fast
            priming of mammalian Atg8 proteins, whereas delipidation is inherently
            slow and
            regulated by all ATG4 homologs
  - term:
      id: GO:0034727
      label: piecemeal microautophagy of the nucleus
    evidence_type: IBA
    original_reference_id: GO_REF:0000033
    review:
      summary: >-
        This IBA annotation suggests ATG4D involvement in piecemeal microautophagy
        of the
        nucleus based on phylogenetic inference. However, direct experimental evidence
        for
        ATG4D specifically in this process in humans is limited.
      action: KEEP_AS_NON_CORE
      reason: >-
        Phylogenetically inferred function. While ATG4 family members are broadly
        involved
        in autophagy processes, piecemeal microautophagy of the nucleus is not a demonstrated
        core function of human ATG4D. The deep research review focuses on macroautophagy
        and
        mitophagy as the main functions.
      additional_reference_ids:
        - file:human/ATG4D/ATG4D-deep-research-falcon.md
  - term:
      id: GO:0035973
      label: aggrephagy
    evidence_type: IBA
    original_reference_id: GO_REF:0000033
    review:
      summary: >-
        ATG4D is involved in aggrephagy (selective autophagy of protein aggregates)
        through
        its general role in autophagosome biogenesis and ATG8 processing. The SQSTM1/p62
        pathway for aggregate clearance requires functional ATG8 lipidation/delipidation.
      action: KEEP_AS_NON_CORE
      reason: >-
        This is a consequence of ATG4D's core autophagy function rather than a specific
        evolved role in aggrephagy. ATG4D enables general autophagy which includes
        aggrephagy, but is not specifically adapted for this selective autophagy pathway.
      supported_by:
        - reference_id: PMID:30661429
          supporting_text: >-
            Cells lacking ATG4B also showed an accumulation of SQSTM1 and a reduced
            clearance
            of this protein upon Torin1 treatment, suggestive of an autophagy defect
  - term:
      id: GO:0005737
      label: cytoplasm
    evidence_type: IEA
    original_reference_id: GO_REF:0000044
    review:
      summary: >-
        IEA annotation confirming cytoplasmic localization based on UniProt subcellular
        location mapping. Consistent with the IBA annotation for the same term.
      action: ACCEPT
      reason: >-
        Redundant with IBA annotation but correct. Cytoplasm is the primary location
        where
        ATG4D functions in ATG8 delipidation on autophagosomal membranes.
      additional_reference_ids:
        - file:human/ATG4D/ATG4D-deep-research-falcon.md
  - term:
      id: GO:0005759
      label: mitochondrial matrix
    evidence_type: IEA
    original_reference_id: GO_REF:0000044
    review:
      summary: >-
        The caspase-3 cleaved form of ATG4D (DeltaN63) localizes to the mitochondrial
        matrix
        after cleavage at DEVD63 exposes a cryptic mitochondrial targeting sequence.
        This
        is not the primary localization of full-length ATG4D.
      action: KEEP_AS_NON_CORE
      reason: >-
        Conditional localization dependent on caspase-3 cleavage during stress/apoptosis.
        The full-length protein is cytoplasmic; only the processed mitochondrial form
        enters the matrix. This represents a secondary, stress-induced localization.
      additional_reference_ids:
        - file:human/ATG4D/ATG4D-deep-research-falcon.md
  - term:
      id: GO:0006508
      label: proteolysis
    evidence_type: IEA
    original_reference_id: GO_REF:0000043
    review:
      summary: >-
        ATG4D is a cysteine protease that performs proteolytic cleavage of ATG8-family
        proteins. This is a broad parent term of the more specific peptidase activities.
      action: ACCEPT
      reason: >-
        Correct but general. ATG4D performs proteolysis as part of its ATG8 priming
        and delipidation functions. More specific terms like cysteine-type peptidase
        activity are more informative.
      supported_by:
        - reference_id: PMID:21177865
          supporting_text: >-
            The Atg4 cysteine proteases are required for processing Atg8 for the latter
            to be conjugated to phosphatidylethanolamine on autophagosomal membranes
  - term:
      id: GO:0006914
      label: autophagy
    evidence_type: IEA
    original_reference_id: GO_REF:0000043
    review:
      summary: >-
        ATG4D is a core autophagy protein involved in both priming and delipidation
        of
        ATG8-family proteins, essential steps in autophagosome biogenesis and maturation.
      action: ACCEPT
      reason: >-
        Core function. ATG4D is an essential component of the autophagy machinery,
        with particular importance for delipidation activity in mammalian cells.
      supported_by:
        - reference_id: PMID:30661429
          supporting_text: >-
            The cysteine protease Atg4 is thought to regulate Atg8 lipidation through
            2 processing steps
  - term:
      id: GO:0006915
      label: apoptotic process
    evidence_type: IEA
    original_reference_id: GO_REF:0000043
    review:
      summary: >-
        This annotation derives from the UniProt Apoptosis keyword, assigned because
        ATG4D
        is cleaved by caspase-3 during apoptosis and the cleaved form translocates
        to
        mitochondria. However, the core evolved function of ATG4D is autophagy, not
        apoptosis.
      action: MARK_AS_OVER_ANNOTATED
      reason: >-
        OVER-ANNOTATION. ATG4D's core function is autophagy (LC3/GABARAP processing).
        The apoptosis connection is secondary - ATG4D is a substrate of caspase-3
        rather
        than having an evolved apoptotic function. The keyword mapping has led to
        over-interpretation. Autophagy and apoptosis have crosstalk, but ATG4D's primary
        role is autophagosome biogenesis through ATG8 delipidation, not apoptosis
        execution.
      additional_reference_ids:
        - file:human/ATG4D/ATG4D-deep-research-falcon.md
  - term:
      id: GO:0008233
      label: peptidase activity
    evidence_type: IEA
    original_reference_id: GO_REF:0000043
    review:
      summary: >-
        ATG4D has peptidase activity as a member of the peptidase C54 family. This
        is a
        correct but general annotation; more specific terms exist.
      action: ACCEPT
      reason: >-
        Correct parent term. ATG4D is a peptidase that cleaves ATG8-family proteins.
        More specific terms (cysteine-type endopeptidase activity) are more informative
        but this annotation is not incorrect.
      supported_by:
        - reference_id: PMID:21177865
          supporting_text: >-
            The Atg4 cysteine proteases are required for processing Atg8
  - term:
      id: GO:0008234
      label: cysteine-type peptidase activity
    evidence_type: IEA
    original_reference_id: GO_REF:0000120
    review:
      summary: >-
        ATG4D is a cysteine-type peptidase of the C54 family. This activity is demonstrated
        by kinetic studies showing cleavage of ATG8 substrates.
      action: ACCEPT
      reason: >-
        Core molecular function. ATG4D uses an active site cysteine (Cys144) for catalysis,
        characteristic of papain-like cysteine proteases.
      supported_by:
        - reference_id: PMID:21177865
          supporting_text: >-
            The Atg4 cysteine proteases are required for processing Atg8
  - term:
      id: GO:0015031
      label: protein transport
    evidence_type: IEA
    original_reference_id: GO_REF:0000043
    review:
      summary: >-
        This annotation likely derives from the UniProt Protein transport keyword.
        ATG4D
        has an indirect role in protein transport through autophagy-mediated processes,
        but this is not its primary function.
      action: MARK_AS_OVER_ANNOTATED
      reason: >-
        Indirect and tangential. While autophagy involves membrane trafficking, ATG4D's
        function is specifically ATG8 processing, not general protein transport. The
        keyword mapping has led to a non-specific annotation that doesn't reflect
        the
        core molecular function of ATG4D.
      additional_reference_ids:
        - file:human/ATG4D/ATG4D-deep-research-falcon.md
  - term:
      id: GO:0016787
      label: hydrolase activity
    evidence_type: IEA
    original_reference_id: GO_REF:0000043
    review:
      summary: >-
        ATG4D is a hydrolase that cleaves peptide bonds in ATG8 proteins and hydrolyzes
        the amide bond between ATG8 and PE/PS during delipidation.
      action: ACCEPT
      reason: >-
        Correct broad parent term. ATG4D performs hydrolysis of both peptide bonds
        (priming) and ATG8-lipid conjugates (delipidation).
      supported_by:
        - reference_id: PMID:30661429
          supporting_text: >-
            Atg4 can hydrolyze the amide linkage between Atg8 and PE in a delipidation/deconjugation
            step that recycles free Atg8
  - term:
      id: GO:0019786
      label: protein-phosphatidylethanolamide deconjugating activity
    evidence_type: IEA
    original_reference_id: GO_REF:0000002
    review:
      summary: >-
        Duplicate annotation of delipidation activity from InterPro mapping. ATG4D
        removes
        PE from ATG8-PE conjugates as a major in vivo function.
      action: ACCEPT
      reason: >-
        Core molecular function. This IEA annotation is redundant with the IBA annotation
        but both correctly capture ATG4D's delipidation activity, which is its predominant
        function in cells.
      supported_by:
        - reference_id: PMID:29458288
          supporting_text: >-
            Delipidation of mammalian Atg8-family proteins by each of the four ATG4
            proteases
  - term:
      id: GO:0005739
      label: mitochondrion
    evidence_type: IDA
    original_reference_id: GO_REF:0000052
    review:
      summary: >-
        Mitochondrial localization based on immunofluorescence data (HPA). The caspase-cleaved
        form of ATG4D (DeltaN63) translocates to mitochondria where it associates
        with
        cardiolipin and affects mitochondrial ultrastructure.
      action: KEEP_AS_NON_CORE
      reason: >-
        Conditional localization. Full-length ATG4D is cytoplasmic; only the caspase-3
        processed form localizes to mitochondria during stress conditions. This is
        a
        secondary function rather than the primary site of ATG4D action.
      additional_reference_ids:
        - file:human/ATG4D/ATG4D-deep-research-falcon.md
  - term:
      id: GO:0000423
      label: mitophagy
    evidence_type: IMP
    original_reference_id: PMID:33773106
    review:
      summary: >-
        Experimental evidence from mutant phenotype showing ATG4D involvement in mitophagy.
        ATG4D promotes phagophore growth during mitophagy by regulating ATG9A trafficking
        to mitochondria.
      action: ACCEPT
      reason: >-
        Direct experimental evidence. ATG4D has a specific role in mitophagy beyond
        general
        autophagy, involving regulation of ATG9A vesicle trafficking to mitochondria
        and
        promoting phagophore-ER contacts during mitophagy.
      supported_by:
        - reference_id: PMID:33773106
          supporting_text: >-
            The sequestration of damaged mitochondria within double-membrane structures
            termed autophagosomes is a key step of PINK1/Parkin mitophagy
  - term:
      id: GO:0006914
      label: autophagy
    evidence_type: IDA
    original_reference_id: PMID:30661429
    review:
      summary: >-
        Direct experimental evidence for ATG4D role in autophagy from comprehensive
        analysis
        of ATG4 isoform redundancy. ATG4D contributes to LC3/GABARAP processing in
        cells.
      action: ACCEPT
      reason: >-
        Core function with strong experimental support. ATG4D contributes to priming
        of GABARAP isoforms and is a major delipidating enzyme (PMID:30661429).
      supported_by:
        - reference_id: PMID:30661429
          supporting_text: >-
            By further depletion of ATG4 isoforms, we discover that ATG4A, ATG4C and
            ATGD all
            contribute to the remaining processing activity and thus show overlapping
            redundancy
            in cells
  - term:
      id: GO:0008234
      label: cysteine-type peptidase activity
    evidence_type: IDA
    original_reference_id: PMID:30661429
    review:
      summary: >-
        Direct experimental demonstration of ATG4D peptidase activity in cells. Loss
        of
        ATG4D (with other ATG4s) affects LC3/GABARAP priming.
      action: ACCEPT
      reason: >-
        Core molecular function experimentally demonstrated. ATG4D contributes to
        LC3/GABARAP processing in cells, particularly GABARAPL1 and GABARAPL2.
      supported_by:
        - reference_id: PMID:30661429
          supporting_text: >-
            All human ATG4 isoforms contribute to the priming of LC3/GABARAP isoforms,
            because complete loss of LC3/GABARAP priming and lipidation in cells could
            only be achieved upon loss of all ATG4 isoforms
  - term:
      id: GO:0034497
      label: protein localization to phagophore assembly site
    evidence_type: IMP
    original_reference_id: PMID:33773106
    review:
      summary: >-
        ATG4D promotes protein localization to the phagophore assembly site by regulating
        ATG9A vesicle trafficking. This is a protease-independent function.
      action: ACCEPT
      reason: >-
        Experimentally demonstrated function. ATG4D has a non-canonical role in promoting
        ATG9A trafficking to the phagophore assembly site, independent of its protease
        activity (PMID:33773106).
      supported_by:
        - reference_id: PMID:33773106
          supporting_text: >-
            ATG4 proximity networks reveal a role for ATG4s and their proximity partners,
            including the immune-disease protein LRBA, in ATG9A vesicle trafficking
            to
            mitochondria
  - term:
      id: GO:0051697
      label: protein delipidation
    evidence_type: IDA
    original_reference_id: PMID:29458288
    review:
      summary: >-
        ATG4D catalyzes protein delipidation, removing ATG8-family proteins from membrane
        lipids. In reconstituted delipidation assays, all four ATG4 proteases can
        be
        activated to similar activities on lipid-attached substrates.
      action: ACCEPT
      reason: >-
        Core molecular function. ATG4D is the predominant delipidating enzyme for
        LC3B
        in cells, more important for delipidation than for priming (PMID:29458288).
      supported_by:
        - reference_id: PMID:29458288
          supporting_text: >-
            In a fully reconstituted delipidation assay, we establish that the physical
            anchoring of mammalian Atg8-family proteins in the membrane dramatically
            shifts
            the way ATG4 proteases recognize these substrates
  - term:
      id: GO:0006508
      label: proteolysis
    evidence_type: IDA
    original_reference_id: PMID:21177865
    review:
      summary: >-
        ATG4D performs proteolysis of ATG8 substrates. Kinetic analysis demonstrated
        proteolytic activity against LC3B and GABARAPL2, though with lower activity
        than ATG4B.
      action: ACCEPT
      reason: >-
        Core function directly demonstrated. ATG4D cleaves ATG8 substrates, with Km
        values
        of 13.1 uM for MAP1LC3B and 7.2 uM for GABARAPL2 (PMID:21177865).
      supported_by:
        - reference_id: PMID:21177865
          supporting_text: >-
            The present study provided the first detailed kinetics analysis of all
            four
            Atg4 homologues against four representative Atg8 homologues
  - term:
      id: GO:0008234
      label: cysteine-type peptidase activity
    evidence_type: IDA
    original_reference_id: PMID:21177865
    review:
      summary: >-
        ATG4D has cysteine-type peptidase activity demonstrated by kinetic analysis.
        Activity is inhibited by N-ethylmaleimide, confirming the cysteine-dependent
        mechanism.
      action: ACCEPT
      reason: >-
        Core molecular function with biochemical characterization. ATG4D uses an active
        site cysteine for catalysis, characteristic of C54 family proteases (PMID:21177865).
      supported_by:
        - reference_id: PMID:21177865
          supporting_text: >-
            The Atg4 cysteine proteases are required for processing Atg8 for the latter
            to be conjugated to phosphatidylethanolamine on autophagosomal membranes
core_functions:
  - description: >-
      ATG4D's primary molecular function is delipidation of ATG8-PE conjugates. While
      it has weak priming activity compared to ATG4B, ATG4D is the major delipidating
      enzyme in mammalian cells, particularly for LC3B.
    molecular_function:
      id: GO:0019786
      label: protein-phosphatidylethanolamide deconjugating activity
    directly_involved_in:
      - id: GO:0006914
        label: autophagy
      - id: GO:0000423
        label: mitophagy
    locations:
      - id: GO:0005737
        label: cytoplasm
    supported_by:
      - reference_id: PMID:29458288
        supporting_text: >-
          all 4 ATG4 proteases can be activated to similar enzymatic activities on
          lipid-attached substrates
      - reference_id: file:human/ATG4D/ATG4D-deep-research-falcon.md
        supporting_text: >-
          ATG4D constitutes the major protein for the delipidation activity, while
          it promotes weak proteolytic activation of ATG8 proteins
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:0000120
    title: Combined Automated Annotation using Multiple IEA Methods
    findings: []
  - id: PMID:21177865
    title: Kinetics comparisons of mammalian Atg4 homologues indicate selective 
      preferences toward diverse Atg8 substrates.
    findings: []
  - id: PMID:29458288
    title: Delipidation of mammalian Atg8-family proteins by each of the four 
      ATG4 proteases.
    findings: []
  - id: PMID:30661429
    title: Redundancy of human ATG4 protease isoforms in autophagy and 
      LC3/GABARAP processing revealed in cells.
    findings: []
  - id: PMID:33773106
    title: ATG4 family proteins drive phagophore growth independently of the 
      LC3/GABARAP lipidation system.
    findings: []
  - id: file:human/ATG4D/ATG4D-deep-research-falcon.md
    title: Deep research review of ATG4D function
    findings: []