DFFA, also called DFF45 or ICAD, is the regulatory subunit of DNA fragmentation factor. Its long isoform promotes productive folding of DFFB/CAD and then inhibits the mature nuclease in a heterodimeric complex. Executioner-caspase cleavage of DFFA removes this restraint, allowing CAD to fragment chromosomal DNA. DFFA-containing complexes occupy cytosolic and nuclear or chromatin-associated pools; their distribution and processing vary with cell type. Folding and nuclease inhibition are separable activities, and shorter isoforms differ in chaperone capacity.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000785 chromatin | IBA GO_REF:0000033 | ACCEPT | Summary: DFFA is active with DFFB on chromatin as part of the DNA fragmentation factor complex. Reason: The DFFA:DFFB complex can associate with chromatin, and DFFA inhibition of DFFB is a core activity that is relieved at chromatin during apoptotic DNA fragmentation. The DFFA-specific PAINT node transfers the same chromatin active-site placement within the ICAD/DFF45 subfamily. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN002642559 SUPPORTS TRANSFER DFFA-specific node; human DFFA is itself an experimental source for the chromatin placement. Supporting Evidence: PMID:15572351 transiently expressed DFF associates with chromatin |
| GO:0000785 chromatin | IDA PMID:19882353 Histone H1 subtype preferences of DFF40 and possible nuclear... | ACCEPT | Summary: DFFA/DFF45 was detected with DFFB/DFF40 in the chromatin-enriched fraction of NB4 cells. Reason: The cited study supports chromatin localization of the DFF40:DFF45 heterodimer in cells. The nuclease subunit mediates the DNA interaction, while the DFFA subunit remains in the complex and inhibits cleavage until caspase processing. Supporting Evidence: PMID:19882353 the heterodimer, DFF40-DFF45, is localized to the |
| GO:0005515 protein binding | IPI PMID:11371636 Solution structure of DFF40 and DFF45 N-terminal domain comp... | MODIFY | Summary: The DFFA-DFFB interaction has specific inhibitor and chaperone functions. Reason: The small-scale NMR and biochemical study established the DFF45:DFF40 interface and reported that DFF45 is required for DFF40 folding and inhibits DFF40 nuclease activity. Replace uninformative generic binding with the inhibitor activity; the folding-chaperone function is added as a separate proposed annotation. Proposed replacements: deoxyribonuclease inhibitor activity Supporting Evidence: PMID:11371636 Expression and folding of DFF40 require the presence of DFF45, |
| GO:0005515 protein binding | IPI PMID:19882353 Histone H1 subtype preferences of DFF40 and possible nuclear... | REMOVE | Summary: The DFF40-DFF45 heterodimer was localized to chromatin rather than functionally characterized as a new DFFA binding activity. Reason: This row records the known DFFB interaction from a chromatin-localization study. That localization is already captured by the chromatin annotation, while the generic protein-binding row adds no specific DFFA molecular function beyond the established inhibitor/chaperone activities. Supporting Evidence: PMID:19882353 the heterodimer, DFF40-DFF45, is localized to the |
| GO:0005515 protein binding | IPI PMID:22727028 Identification of ICAD-derived peptides capable of inhibitin... | MODIFY | Summary: ICAD-derived peptides inhibit mouse CAD/Dffb, supporting DFFA-family DNase inhibitor activity. Reason: The peptide-mapping study used ICAD-derived sequences to identify regions that inhibit CAD nuclease activity. Replace generic protein binding with deoxyribonuclease inhibitor activity; the mechanism is inhibition of CAD/DFFB, not a standalone binding function. Proposed replacements: deoxyribonuclease inhibitor activity Supporting Evidence: PMID:22727028 capable of efficiently and specifically inhibiting CAD activity in solution. |
| GO:0005515 protein binding | IPI PMID:25277244 The functional landscape of Hsp27 reveals new cellular proce... | REMOVE | Summary: The HSPB1/Hsp27 interaction row does not define a DFFA activity. Reason: This HSPB1-centered interactome study reported a physical interaction with DFFA but does not establish a specific molecular function for DFFA or place HSPB1 in the ICAD/CAD DNA-fragmentation module. Generic protein binding is therefore not informative here. |
| GO:0005515 protein binding | IPI PMID:32296183 A reference map of the human binary protein interactome. | REMOVE | Summary: The HuRI DFFA-DFFB edge redetects the known DFF interaction without adding a new DFFA function. Reason: High-throughput binary interaction mapping can recover known partners, but this generic DFFB edge does not add functional information beyond the focused DFF40:DFF45 inhibitor/chaperone literature. |
| GO:0005515 protein binding | IPI PMID:33961781 Dual proteome-scale networks reveal cell-specific remodeling... | REMOVE | Summary: The BioPlex DFFA-DFFB edge redetects the known DFF interaction without adding a new DFFA function. Reason: Affinity-purification interactome mapping can recover known complex partners, but this generic DFFB edge does not add functional information beyond the focused DFF40:DFF45 inhibitor/chaperone literature. |
| GO:0005515 protein binding | IPI PMID:33961781 Dual proteome-scale networks reveal cell-specific remodeling... | REMOVE | Summary: The TSPYL4 BioPlex edge is not a demonstrated DFFA molecular function. Reason: The BioPlex screen is a high-throughput AP-MS resource. Without follow-up evidence that TSPYL4 changes ICAD/CAD folding, inhibition, or apoptotic DNA fragmentation, this generic binding row should not be retained as DFFA function. |
| GO:0005515 protein binding | IPI PMID:40205054 Multimodal cell maps as a foundation for structural and func... | REMOVE | Summary: The multimodal cell-map DFFA-DFFB edge redetects the known DFF interaction without adding a new DFFA function. Reason: The Cell Map interaction is consistent with the DFF complex but does not define a more specific function than generic protein binding. Focused DFF40:DFF45 studies already capture the inhibitor/chaperone biology. |
| GO:0005634 nucleus | IDA PMID:15572351 Interaction of DNA fragmentation factor (DFF) with DNA revea... | ACCEPT | Summary: DFFA can localize to the nucleus as part of the DFF complex. Reason: DFF45/ICAD forms a complex with DFF40/CAD and DFF has been observed associated with DNA/chromatin in transfected cells, consistent with nuclear residence of a DFF pool. Supporting Evidence: PMID:15572351 DNA fragmentation factor (DFF) is a complex of the DNase DFF40 |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-211186 | ACCEPT | Summary: Reactome places DFFA/DFF45 in the nucleoplasm as part of nuclear DFF import, cleavage, and release of DFF40. Reason: DFFA participates in the DFF complex during importin-mediated nuclear import and caspase-3-dependent cleavage in the nucleus. The importin-dissociation row is inferred from the general importin-alpha/beta Ran:GTP mechanism, but it remains consistent with DFF nuclear import. Supporting Evidence: Reactome:R-HSA-211186 Caspase-3 cleaves DFF45 between residues 224,225. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-211190 | ACCEPT | Summary: Reactome places DFFA/DFF45 in the nucleoplasm as part of nuclear DFF import, cleavage, and release of DFF40. Reason: DFFA participates in the DFF complex during importin-mediated nuclear import and caspase-3-dependent cleavage in the nucleus. The importin-dissociation row is inferred from the general importin-alpha/beta Ran:GTP mechanism, but it remains consistent with DFF nuclear import. Supporting Evidence: Reactome:R-HSA-211190 Caspase-3 cleaves the DFF45 subunit of the DFF45:DFF40 complex |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-211206 | ACCEPT | Summary: Reactome places DFFA/DFF45 in the nucleoplasm as part of nuclear DFF import, cleavage, and release of DFF40. Reason: DFFA participates in the DFF complex during importin-mediated nuclear import and caspase-3-dependent cleavage in the nucleus. The importin-dissociation row is inferred from the general importin-alpha/beta Ran:GTP mechanism, but it remains consistent with DFF nuclear import. Supporting Evidence: Reactome:R-HSA-211206 DFF associated with alpha-importin:beta-importin is translocated to the nucleus |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-211207 | ACCEPT | Summary: Reactome places DFFA/DFF45 in the nucleoplasm as part of nuclear DFF import, cleavage, and release of DFF40. Reason: DFFA participates in the DFF complex during importin-mediated nuclear import and caspase-3-dependent cleavage in the nucleus. The importin-dissociation row is inferred from the general importin-alpha/beta Ran:GTP mechanism, but it remains consistent with DFF nuclear import. Supporting Evidence: Reactome:R-HSA-211207 the fragments of DFF45 dissociate from DFF40 |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-9029667 | ACCEPT | Summary: Reactome places DFFA/DFF45 in the nucleoplasm as part of nuclear DFF import, cleavage, and release of DFF40. Reason: DFFA participates in the DFF complex during importin-mediated nuclear import and caspase-3-dependent cleavage in the nucleus. The importin-dissociation row is inferred from the general importin-alpha/beta Ran:GTP mechanism, but it remains consistent with DFF nuclear import. Supporting Evidence: Reactome:R-HSA-9029667 Dissociation of the DFF complex from the importin alpha/beta complex is therefore assumed. |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | MODIFY | Summary: A cytosolic DFF pool supports a more precise location. Reason: Biochemical purification and full PMID:22253444 resolve a cytosolic pool, already represented in this review; refine cytoplasm to cytosol without excluding the independently supported nuclear pool. Proposed replacements: cytosol Supporting Evidence: PMID:9108473 purified from HeLa cytosol |
| GO:0005829 cytosol | IDA GO_REF:0000052 | ACCEPT | Summary: The Human Protein Atlas immunofluorescence pipeline supports cytosolic DFFA localization. Reason: Cytosolic localization is consistent with the original biochemical purification of DFF from HeLa cytosol and with later cell-biological evidence that ICAD processing can preferentially occur in the cytosolic fraction. Supporting Evidence: PMID:9108473 purified from HeLa cytosol |
| GO:0005829 cytosol | IDA PMID:22253444 Apoptotic DNA degradation into oligonucleosomal fragments, b... | ACCEPT | Summary: ICAD/DFFA is present in cytosolic fractions in neuroblastoma-derived cells. Reason: The cited study directly compared cytosolic and nuclear ICAD/DFF40 pools and found that cytosolic processing of ICAD permits CAD movement into a chromatin-enriched fraction during staurosporine-induced apoptosis. Supporting Evidence: PMID:22253444 the preferential processing of ICAD in the cytosolic fraction |
| GO:0005829 cytosol | IDA PMID:9108473 DFF, a heterodimeric protein that functions downstream of ca... | ACCEPT | Summary: DFF was originally purified from HeLa cytosol. Reason: Cytosolic localization is correct for the DFF complex: the original purification identified a caspase-3-activated cytosolic DFF heterodimer that induces DNA fragmentation in nuclei. Supporting Evidence: PMID:9108473 purified from HeLa cytosol |
| GO:0005829 cytosol | TAS Reactome:R-HSA-211191 | ACCEPT | Summary: Reactome places DFF40:DFF45 in the cytosol for importin-alpha/beta binding before nuclear import. Reason: DFF40:DFF45 can occupy a cytosolic pool and bind importin-alpha/beta before nuclear translocation, matching biochemical data that cytosolic ICAD processing releases CAD for chromatin translocation in some apoptotic cells. Supporting Evidence: Reactome:R-HSA-211191 The translocation of the DFF complex from the cytoplasm to the nucleus is mediated by the importin alfa/beta heterodimer. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-211206 | ACCEPT | Summary: Reactome places DFF40:DFF45 in the cytosol immediately before importin-dependent nuclear translocation. Reason: DFF40:DFF45 can occupy a cytosolic pool that undergoes importin-alpha/beta-dependent nuclear import during the apoptotic DNA-fragmentation pathway. Supporting Evidence: Reactome:R-HSA-211206 DFF associated with alpha-importin:beta-importin is translocated to the nucleus |
| GO:0005829 cytosol | TAS Reactome:R-HSA-211224 | ACCEPT | Summary: Reactome places DFFA:DFFB association in the cytosol. Reason: DFFA and DFFB form the inactive DFF complex; Reactome correctly locates DFF45 chaperone/inhibitor complex formation with DFF40 before DFF moves through the pathway to CAD activation. Supporting Evidence: Reactome:R-HSA-211224 DFF45 (ICAD) appears to act as a chaperone for DFF40 (CAD) during its synthesis |
| GO:0006309 apoptotic DNA fragmentation | IEA GO_REF:0000002 | MODIFY | Summary: DFFA regulates apoptotic DNA fragmentation by folding and inhibiting CAD/DFFB before caspase cleavage releases the nuclease. Reason: InterPro IPR017299 is DFF45/ICAD-specific, but DFFA itself is not the DNA endonuclease; it chaperones DFFB/CAD folding and blocks CAD nuclease activity until caspase-3 cleavage removes the inhibitor. The DFFA-side process should therefore be negative regulation of apoptotic DNA fragmentation rather than DNA fragmentation itself. Proposed replacements: negative regulation of apoptotic DNA fragmentation Supporting Evidence: PMID:11371636 which also acts as a nuclease inhibitor before DFF40 activation |
| GO:0006915 apoptotic process | IBA GO_REF:0000033 | MODIFY | Summary: Top-level apoptotic process is true but too broad for DFFA/ICAD. Reason: DFFA acts specifically in CAD-dependent apoptotic DNA fragmentation as a folding chaperone and DNase inhibitor, so the generic apoptotic-process assertion should be narrowed to negative regulation of apoptotic DNA fragmentation. The broad PAINT donor set includes CIDEA/CIDEB/CIDEC family members as well as DFFA, while the DFFA-specific node supports the narrower term. This refinement follows DFFA-specific inhibitor/chaperone biology, not the number or diversity of extant PAINT donors. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN000959605 SUPPORTS SOURCE BUT NOT TARGET Broad PTHR12306 CIDE-family node; DFFA needs a CAD/DFFB-specific DNA-fragmentation assertion rather than top-level apoptotic process. Proposed replacements: negative regulation of apoptotic DNA fragmentation Supporting Evidence: PMID:11371636 which also acts as a nuclease inhibitor before DFF40 activation |
| GO:0006915 apoptotic process | IEA GO_REF:0000002 | MODIFY | Summary: The CIDE-N InterPro mapping to top-level apoptotic process is too broad for DFFA/ICAD. Reason: IPR003508 marks the CIDE-N domain shared by DFFA and CIDE lipid-transferase paralogs. For DFFA, the evidence-supported process is the narrower negative regulation of apoptotic DNA fragmentation through ICAD/CAD complex formation. This refinement follows DFFA-specific inhibitor/chaperone biology, not the number or diversity of extant PAINT donors. Proposed replacements: negative regulation of apoptotic DNA fragmentation |
| GO:0032991 protein-containing complex | IDA PMID:11371636 Solution structure of DFF40 and DFF45 N-terminal domain comp... | ACCEPT | Summary: DFFA is a subunit of the functional DFF40-DFF45 heterodimer. Reason: Full PMID:11371636 directly resolves the human heterodimer. Stable membership in this inhibitory/chaperoning assembly is central to DFFA biology. The general complex term is retained at the available ontology resolution rather than calling core assembly membership peripheral. Supporting Evidence: PMID:11371636 The solution structure of the heterodimeric complex between NTDs of DFF40 and DFF45 |
| GO:0042981 regulation of apoptotic process | IEA GO_REF:0000117 | MODIFY | Summary: ARBA mapped DFFA to a generic apoptosis-regulation parent. Reason: Regulation of apoptotic process is much broader than the direct DFFA role. The DFF45/ICAD mechanism is sequestration of CAD/DFF40 nuclease activity until caspase cleavage, best represented as negative regulation of apoptotic DNA fragmentation. Proposed replacements: negative regulation of apoptotic DNA fragmentation |
| GO:0060703 deoxyribonuclease inhibitor activity | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro DFF45-family membership supports the core DFFB/CAD inhibitor activity. Reason: IPR017299 is DFF45/ICAD-specific, and DFFA inhibition of CAD/DFF40 is the central molecular function of this family. Supporting Evidence: PMID:19944011 DFF40 exists as a heterodimer with DFF45 |
| GO:0060703 deoxyribonuclease inhibitor activity | IMP PMID:19944011 Fifty C-terminal amino acid residues are necessary for the c... | ACCEPT | Summary: The C terminus is needed for DFFA chaperone activity but not for CAD inhibition. Reason: The mutational/biochemical study dissected two DFFA activities and explicitly found that the C-terminal helix is needed for DFF45 chaperone activity but not for inhibition of DFF40, supporting deoxyribonuclease inhibitor activity as a separable molecular function. Supporting Evidence: PMID:19944011 necessary for the chaperone activity of DFF45, but not for inhibition of DFF40 |
| GO:1900118 negative regulation of execution phase of apoptosis | IBA GO_REF:0000033 | ACCEPT | Summary: DFFA negatively regulates apoptotic execution by restraining CAD-mediated DNA fragmentation. Reason: This DFFA-specific PAINT annotation is biologically sound: ICAD/DFFA prevents premature CAD nuclease activity, and caspase cleavage of ICAD releases the active CAD nuclease during the execution phase. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN002642559 SUPPORTS TRANSFER DFFA-specific node supports transfer of execution-phase inhibition through the ICAD/DFF45 subfamily. Supporting Evidence: PMID:9108473 Caspase-3 cleaves the 45 kDa subunit at two sites |
| GO:1900118 negative regulation of execution phase of apoptosis | IDA PMID:9564035 CIDE, a novel family of cell death activators with homology ... | ACCEPT | Summary: DFF45 can suppress CIDE-enhanced apoptotic execution phenotypes. Reason: The CIDE discovery paper used an overexpression system, but it is consistent with DFF45 acting as an inhibitor in the execution portion of the DFF/CIDE family death program. Supporting Evidence: PMID:9564035 CD95/Fas-mediated apoptosis was enhanced by CIDEs but inhibited by DFF45. |
| GO:1902511 negative regulation of apoptotic DNA fragmentation | IBA GO_REF:0000033 | ACCEPT | Summary: DFFA negatively regulates CAD-mediated apoptotic DNA fragmentation. Reason: This DFFA-specific PAINT annotation captures the core ICAD/DFF45 role: DFFA binds and inhibits CAD/DFFB until caspase cleavage permits CAD activation. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN002642559 SUPPORTS TRANSFER DFFA-specific node supports the inherited CAD/DFFB inhibitory role. Supporting Evidence: PMID:11371636 which also acts as a nuclease inhibitor before DFF40 activation |
| GO:1902511 negative regulation of apoptotic DNA fragmentation | IDA PMID:11371636 Solution structure of DFF40 and DFF45 N-terminal domain comp... | ACCEPT | Summary: DFFA-DFFB binding inhibits CAD/DFFB and thereby prevents premature apoptotic DNA fragmentation. Reason: The NMR study established the DFF40:DFF45 N-terminal complex and described DFF45 both as the folding requirement for DFF40 and as its nuclease inhibitor before executioner-caspase activation. Supporting Evidence: PMID:11371636 which also acts as a nuclease inhibitor before DFF40 activation |
| GO:1902511 negative regulation of apoptotic DNA fragmentation | IDA PMID:15572351 Interaction of DNA fragmentation factor (DFF) with DNA revea... | ACCEPT | Summary: DFF45 inhibition of DFF40 permits DFF to bind DNA without cutting it. Reason: The cited study shows that the DFF complex is competent to bind DNA but catalytically inactive until activation, directly supporting DFFA-mediated negative regulation of CAD-dependent apoptotic DNA cleavage. Supporting Evidence: PMID:15572351 reconstituted DFF is catalytically inactive yet proficient in DNA binding |
| GO:1902511 negative regulation of apoptotic DNA fragmentation | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA correctly mapped DFFA to negative regulation of apoptotic DNA fragmentation. Reason: Despite being electronic, the assertion matches the core ICAD/DFF45 mechanism: DFFA binds CAD/DFFB and inhibits its DNase activity until caspase cleavage releases the active nuclease. Supporting Evidence: PMID:19944011 DFF40 exists as a heterodimer with DFF45 |
| GO:1902511 negative regulation of apoptotic DNA fragmentation | IMP PMID:19944011 Fifty C-terminal amino acid residues are necessary for the c... | ACCEPT | Summary: DFFA C-terminal mutants separate CAD folding from CAD inhibition. Reason: The cited mutational analysis supports negative regulation of apoptotic DNA fragmentation because even DFF45 variants lacking the C-terminal chaperone segment can still inhibit DFF40 nuclease activity. Supporting Evidence: PMID:19944011 necessary for the chaperone activity of DFF45, but not for inhibition of DFF40 |
| GO:0044183 protein folding chaperone | IDA PMID:11371636 Solution structure of DFF40 and DFF45 N-terminal domain comp... | NEW | Summary: DFFA/ICAD is a client-specific folding chaperone for CAD/DFFB. Reason: DFFA is required for productive DFF40/CAD folding and remains complexed with the nuclease as its inhibitor. This is a direct molecular chaperone activity distinct from the DNase-inhibitor activity because DFFA supports formation of active DFF40 before blocking its nuclease activity. Full PMID:11371636 describes mutual N-terminal-domain folding and distinguishes DFFA-mediated folding of the DFFB catalytic domain. This is direct chaperone work, not merely necessity as a caspase substrate. Supporting Evidence: PMID:11371636 Expression and folding of DFF40 require the presence of DFF45, PMID:19944011 necessary for the chaperone activity of DFF45, but not for inhibition of DFF40 |
| GO:0006457 protein folding | IDA PMID:11371636 Solution structure of DFF40 and DFF45 N-terminal domain comp... | NEW | Summary: DFFA/ICAD assists productive folding of CAD/DFFB. Reason: DFFA is required for DFF40/CAD expression and folding before staying bound to the mature nuclease as its inhibitor. This makes DFFA directly involved in folding a specific protein substrate rather than merely binding an assembled DFF40:DFF45 complex. Full PMID:11371636 describes mutual N-terminal-domain folding and distinguishes DFFA-mediated folding of the DFFB catalytic domain. This is direct chaperone work, not merely necessity as a caspase substrate. Supporting Evidence: PMID:11371636 Expression and folding of DFF40 require the presence of DFF45, PMID:19944011 necessary for the chaperone activity of DFF45, but not for inhibition of DFF40 |
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Download this section (compressed HTML)Q: Should GO add a DNA fragmentation factor complex cellular-component term so DFFA and DFFB complex membership can be curated more specifically than protein-containing complex?
Q: Should GO distinguish ICAD-like dedicated nuclease folding chaperones from general protein-folding chaperones, or is GO:0044183 sufficiently expressive for DFFA-assisted CAD folding?
Experiment: Endogenously tag DFFA and DFFB in cells competent and incompetent for DNA laddering, then measure cytosolic, nuclear, and chromatin-associated DFF pools before and after apoptotic stimulation while rescuing with caspase-resistant DFFA and chaperone-defective C-terminal DFFA variants.
Hypothesis: The cytosolic pool of correctly chaperoned DFFA:DFFB is the caspase-accessible source of CAD that translocates to chromatin for oligonucleosomal DNA fragmentation.
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