Ninjurin-1 is a small, widely expressed plasma-membrane protein of four alpha helices: two transmembrane helices (alpha3, alpha4) and two amphipathic helices (alpha1, alpha2) that are sequestered in the bilayer in the resting state. It is the executioner of plasma membrane rupture (PMR), the terminal, active step of lytic cell death. On a death signal, NINJ1 leaves its autoinhibited dimer, exposes the hydrophilic faces of alpha1 and alpha2 and polymerises laterally into branched filaments and rings in the plasma membrane; these amphipathic assemblies cap membrane edges and release membrane fragments, so the membrane is lost rather than merely perforated. PMR discharges damage-associated molecular patterns such as HMGB1 and LDH and thereby amplifies inflammation. NINJ1 acts downstream of gasdermins in pyroptosis and of MLKL in necroptosis, after apoptosis, during ferroptosis and after osmotic swelling, and it can also rupture membranes of glucose-starved macrophages independently of any known cell-death program; how its filaments finish the job - by excising membrane disks or by opening very large pores - is still argued. NINJ1 was originally characterised as a homophilic cell adhesion molecule induced by nerve injury, adhering through a short extracellular N-terminal adhesion motif, and it retains non-lytic activities: it promotes monocyte transendothelial migration, angiogenesis, and, together with ninjurin-2, haematopoietic stem and progenitor cell amplification via canonical WNT signalling, a function that persists in PMR-deficient mutants. A secreted form generated by cleavage has separate chemotactic and anti-inflammatory activity. Whether the adhesive and the membrane-destabilising activities are two uses of the same amphipathic helices, or genuinely separate functions, is unresolved.
Definition: The active, protein-mediated disintegration of the plasma membrane that terminates lytic cell death, resulting in loss of membrane continuity and release of cytoplasmic contents including damage-associated molecular patterns. This is distinct from passive osmotic lysis and occurs downstream of, and separably from, pore formation by gasdermins or MLKL.
Justification: GO currently offers only GO:0019835 cytolysis, which conflates the active terminal step with lysis generally, and no term distinguishes membrane permeabilisation (gasdermin pores) from membrane rupture proper. The distinction is exactly what the NINJ1 literature established, and it is experimentally separable: Ninj1-null cells still die and still form gasdermin pores, but fail to rupture and persist as ballooned corpses.
Parent term: cytolysis
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005886 plasma membrane | IBA GO_REF:0000033 | ACCEPT | Summary: NINJ1 is a multi-pass integral protein of the plasma membrane, and this is where it oligomerises and ruptures the bilayer. Reason: Core location, established by cryo-EM and super-resolution imaging of NINJ1 assemblies in the membranes of dying cells and consistent across the IBA, the UniProt mapping and four independent IDAs. Supporting Evidence: PMID:37198476 Super-resolution microscopy reveals that NINJ1 clusters into structurally diverse assemblies in the membranes of dying cells, in particular large, filamentous assemblies with branched morphology. |
| GO:0019835 cytolysis | IBA GO_REF:0000033 | ACCEPT | Summary: NINJ1 execution of plasma membrane rupture, that is, the loss of membrane integrity and release of cytoplasmic contents at the end of lytic cell death. Reason: The core biological process and the closest term GO currently offers to plasma membrane rupture. Directly demonstrated: Ninj1-null macrophages die but fail to release LDH and HMGB1 and remain as persistent ballooned corpses. Supporting Evidence: PMID:33472215 Ninj1-/- macrophages exhibited impaired PMR in response to diverse inducers of pyroptotic, necrotic and apoptotic cell death, and were unable to release numerous intracellular proteins including HMGB1 (a known DAMP) and LDH (a standard measure of PMR). |
| GO:0098632 cell-cell adhesion mediator activity | IBA GO_REF:0000033 | ACCEPT | Summary: Homophilic trans-interaction between NINJ1 molecules on apposed cell surfaces, mediated by a short extracellular N-terminal adhesion motif. Reason: Retained as a second, genuine core function rather than a historical artefact. The original characterisation used cell-aggregation assays and mapped the adhesive determinant to an 11-residue extracellular region, and competitive peptides from that region block both aggregation and neurite outgrowth; the same motif was later shown to bind endogenous NINJ1 in a pull-down. Independent support that NINJ1 has real non-lytic biology comes from PMR-deficient Ninj1 mutants that nevertheless retain a haematopoietic function. The caveat, recorded rather than hidden: that haematopoietic role was traced to canonical WNT signalling, not to the adhesion motif, so the adhesive activity has not yet been tied to a current mechanism, and the same extracellular helices serve the rupture activity. Supporting Evidence: PMID:9261151 Aggregation assays were used to demonstrate that ninjurin-mediated adhesion requires divalent cations and is an energy-dependent process. PMID:9261151 The critical domain for ninjurin-mediated homophilic adhesion was localized to an 11-residue region (between Pro26 and Asn37) by mutagenesis and by employing synthetic oligopeptides as competitive inhibitors of ninjurin-mediated adhesion. PMID:42481508 Ninj1 deficiency reduced HSPC, neutrophil and erythrocyte numbers, whereas PMR-deficient Ninj1 mutants demonstrated that this function is independent of PMR. |
| GO:0140912 membrane destabilizing activity | IBA GO_REF:0000033 | ACCEPT | Summary: NINJ1 binds the plasma membrane and destroys its integrity: it polymerises into amphipathic filaments whose hydrophilic face caps a membrane edge, so membrane material is shed rather than merely perforated. Reason: The core molecular function, and the single best-evidenced thing NINJ1 does. Supported by six independent IDAs (forward-genetic screen, antibody blockade, two cryo-EM structures, reconstitution with recombinant protein) plus the phylogenetic inference. The mechanistic detail is still argued - Degen et al. and Dai and colleagues describe filaments that cap or solubilise membrane fragments, whereas Sahoo et al. also see less frequent pore-like assemblies - but every model is an instance of this term, so the disagreement is about mechanism, not about whether the activity exists. Supporting Evidence: PMID:37198476 Our data thus suggest that, during lytic cell death, the extracellular Ξ±-helices of NINJ1 insert into the plasma membrane to polymerize NINJ1 monomers into amphipathic filaments that rupture the plasma membrane. PMID:38614101 This structural observation suggests that NINJ1 can form membrane disks, consistent with membrane fragmentation by recombinant NINJ1. PMID:39667936 This structural feature and other evidence point to a PMR mechanism by which NINJ1 filaments wrap around and solubilize membrane fragments and, less frequently, form pores in the plasma membrane. PMID:33472215 Mechanistically, NINJ1 used an evolutionarily conserved extracellular domain for oligomerization and subsequent PMR. |
| GO:0141201 pyroptotic cell death | IBA GO_REF:0000033 | ACCEPT | Summary: NINJ1 carries out the terminal membrane-rupture step of pyroptosis, downstream of gasdermin pore formation. Reason: Best-evidenced of the cell-death contexts in which NINJ1 acts, with four human IDAs and the phylogenetic inference. Worth stating plainly that NINJ1 is not pyroptosis-specific: it performs the same act after apoptosis, in necroptosis and in ferroptosis, and in glucose-starved macrophages it ruptures membranes with no known death program engaged at all. The term is accepted as a genuine context of the one core activity, not as evidence of pathway specificity. Supporting Evidence: PMID:37196676 Here we describe an anti-NINJ1 monoclonal antibody that specifically targets mouse NINJ1 and blocks oligomerization of NINJ1, preventing PMR. PMID:42270656 In glucose-starved macrophages, NINJ1 ruptures membranes independently of known cell death programs. |
| GO:0007155 cell adhesion | IBA GO_REF:0000033 | ACCEPT | Summary: Homophilic adhesion between cells expressing NINJ1. Reason: The biological-process counterpart of the retained cell-cell adhesion mediator activity, supported by aggregation assays in the founding work, by the N-NAM peptide study and by the phylogenetic inference across the ninjurin family including the Drosophila members. Supporting Evidence: PMID:9261151 Aggregation assays were used to demonstrate that ninjurin-mediated adhesion requires divalent cations and is an energy-dependent process. |
| GO:0005576 extracellular region | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Extracellular localisation, mapped from the UniProt subcellular-location vocabulary. Reason: Not an error: UniProt annotates a separate 'Secreted ninjurin-1' chain generated by cleavage, with chemotactic and anti-inflammatory activity. That secreted species is a minor, post-translationally generated form; the protein that carries the characterised functions is the integral plasma-membrane form, so this is retained but not core. |
| GO:0005886 plasma membrane | IEA GO_REF:0000044 | ACCEPT | Summary: NINJ1 is a multi-pass integral protein of the plasma membrane, and this is where it oligomerises and ruptures the bilayer. Reason: Core location, established by cryo-EM and super-resolution imaging of NINJ1 assemblies in the membranes of dying cells and consistent across the IBA, the UniProt mapping and four independent IDAs. Supporting Evidence: PMID:37198476 Super-resolution microscopy reveals that NINJ1 clusters into structurally diverse assemblies in the membranes of dying cells, in particular large, filamentous assemblies with branched morphology. |
| GO:0007155 cell adhesion | IEA GO_REF:0000120 | ACCEPT | Summary: Homophilic adhesion between cells expressing NINJ1. Reason: The biological-process counterpart of the retained cell-cell adhesion mediator activity, supported by aggregation assays in the founding work, by the N-NAM peptide study and by the phylogenetic inference across the ninjurin family including the Drosophila members. Supporting Evidence: PMID:9261151 Aggregation assays were used to demonstrate that ninjurin-mediated adhesion requires divalent cations and is an energy-dependent process. |
| GO:0016020 membrane | IEA GO_REF:0000002 | MODIFY | Summary: Generic membrane localisation from the InterPro signature. Reason: Correct but uninformative. The specific compartment is established experimentally and is already annotated for this gene. Proposed replacements: plasma membrane |
| GO:0042246 tissue regeneration | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: Tissue regeneration, assigned electronically from the ninjurin InterPro signature. Reason: Reflects the gene's discovery as a nerve-injury-induced protein that promotes Schwann-cell maturation and neurite outgrowth. Genuine historical biology and plausibly downstream of the adhesion activity, but a distal organismal outcome, not a molecular role. |
| GO:0097060 synaptic membrane | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Synaptic membrane localisation, transferred from mouse Ninj1 (UniProtKB:O70131) and carried through the UniProt subcellular-location mapping. Reason: UniProt records 'Synaptic cell membrane' for NINJ1 only by similarity to the mouse protein, with no direct human evidence and no mechanism connecting it to either the rupture or the adhesion activity. Retained as a plausible tissue-specific location but not core. |
| GO:0005515 protein binding | IPI PMID:25416956 A proteome-scale map of the human interactome network. | REMOVE | Summary: Bare protein binding from two systematic yeast two-hybrid interactome maps. Reason: Carries no functional information. The interactors are a heterogeneous set of unrelated membrane proteins from proteome-scale binary screens, with no follow-up establishing biological relevance to NINJ1. No more informative molecular function is supportable from this evidence, so the row is removed rather than rewritten. Removal does not imply the reported interaction is false. |
| GO:0005515 protein binding | IPI PMID:32296183 A reference map of the human binary protein interactome. | REMOVE | Summary: Bare protein binding from two systematic yeast two-hybrid interactome maps. Reason: Carries no functional information. The interactors are a heterogeneous set of unrelated membrane proteins from proteome-scale binary screens, with no follow-up establishing biological relevance to NINJ1. No more informative molecular function is supportable from this evidence, so the row is removed rather than rewritten. Removal does not imply the reported interaction is false. |
| GO:0045766 positive regulation of angiogenesis | IEA GO_REF:0000107 | KEEP AS NON CORE | Summary: An N-terminal adhesion-motif peptide that antagonises endogenous NINJ1 promotes endothelial proliferation, migration and tube formation, implying that NINJ1 itself restrains these. Reason: Downstream tissue-level physiology, and note the direction of the underlying experiment: the pro-angiogenic effect was produced by suppressing endogenous NINJ1 with the N-NAM peptide, so 'positive regulation of angiogenesis' is not the most transparent reading of that result. Retained as peripheral rather than corrected, because this is an experimental annotation whose full text was read by the curator and the rat orthology transfer agrees with it. Supporting Evidence: PMID:33028854 We found that N-NAM promotes proliferation, migration, and tube formation of HUVECs and demonstrate that the suppression of endogenous Ninj1 is responsible for the N-NAM-mediated pro-angiogenic effects. |
| GO:0098631 cell adhesion mediator activity | IEA GO_REF:0000107 | MODIFY | Summary: Parent adhesion-mediator term, from the N-NAM peptide study and by orthology from rat Ninj1. Reason: Correct in kind but under-specific. The demonstrated activity is homophilic NINJ1-NINJ1 binding between apposed cells, which the child term states exactly; the parent would also cover adhesion to an extracellular substrate, which has not been shown. Proposed replacements: cell-cell adhesion mediator activity Supporting Evidence: PMID:33028854 Importantly, a pull-down assay revealed a direct binding between exogenously delivered N-NAM and endogenous Ninj1 and it is N-terminal adhesion motif dependent. |
| GO:0097060 synaptic membrane | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Synaptic membrane localisation, transferred from mouse Ninj1 (UniProtKB:O70131) and carried through the UniProt subcellular-location mapping. Reason: UniProt records 'Synaptic cell membrane' for NINJ1 only by similarity to the mouse protein, with no direct human evidence and no mechanism connecting it to either the rupture or the adhesion activity. Retained as a plausible tissue-specific location but not core. |
| GO:0005886 plasma membrane | IDA PMID:39667936 How NINJ1 mediates plasma membrane rupture and why NINJ2 can... | ACCEPT | Summary: NINJ1 is a multi-pass integral protein of the plasma membrane, and this is where it oligomerises and ruptures the bilayer. Reason: Core location, established by cryo-EM and super-resolution imaging of NINJ1 assemblies in the membranes of dying cells and consistent across the IBA, the UniProt mapping and four independent IDAs. Supporting Evidence: PMID:37198476 Super-resolution microscopy reveals that NINJ1 clusters into structurally diverse assemblies in the membranes of dying cells, in particular large, filamentous assemblies with branched morphology. |
| GO:0019835 cytolysis | IDA PMID:39667936 How NINJ1 mediates plasma membrane rupture and why NINJ2 can... | ACCEPT | Summary: NINJ1 execution of plasma membrane rupture, that is, the loss of membrane integrity and release of cytoplasmic contents at the end of lytic cell death. Reason: The core biological process and the closest term GO currently offers to plasma membrane rupture. Directly demonstrated: Ninj1-null macrophages die but fail to release LDH and HMGB1 and remain as persistent ballooned corpses. Supporting Evidence: PMID:33472215 Ninj1-/- macrophages exhibited impaired PMR in response to diverse inducers of pyroptotic, necrotic and apoptotic cell death, and were unable to release numerous intracellular proteins including HMGB1 (a known DAMP) and LDH (a standard measure of PMR). |
| GO:0140912 membrane destabilizing activity | IDA PMID:39667936 How NINJ1 mediates plasma membrane rupture and why NINJ2 can... | ACCEPT | Summary: NINJ1 binds the plasma membrane and destroys its integrity: it polymerises into amphipathic filaments whose hydrophilic face caps a membrane edge, so membrane material is shed rather than merely perforated. Reason: The core molecular function, and the single best-evidenced thing NINJ1 does. Supported by six independent IDAs (forward-genetic screen, antibody blockade, two cryo-EM structures, reconstitution with recombinant protein) plus the phylogenetic inference. The mechanistic detail is still argued - Degen et al. and Dai and colleagues describe filaments that cap or solubilise membrane fragments, whereas Sahoo et al. also see less frequent pore-like assemblies - but every model is an instance of this term, so the disagreement is about mechanism, not about whether the activity exists. Supporting Evidence: PMID:37198476 Our data thus suggest that, during lytic cell death, the extracellular Ξ±-helices of NINJ1 insert into the plasma membrane to polymerize NINJ1 monomers into amphipathic filaments that rupture the plasma membrane. PMID:38614101 This structural observation suggests that NINJ1 can form membrane disks, consistent with membrane fragmentation by recombinant NINJ1. PMID:39667936 This structural feature and other evidence point to a PMR mechanism by which NINJ1 filaments wrap around and solubilize membrane fragments and, less frequently, form pores in the plasma membrane. PMID:33472215 Mechanistically, NINJ1 used an evolutionarily conserved extracellular domain for oligomerization and subsequent PMR. |
| GO:0005886 plasma membrane | IDA PMID:38396301 NINJ1 induces plasma membrane rupture and release of damage-... | ACCEPT | Summary: NINJ1 is a multi-pass integral protein of the plasma membrane, and this is where it oligomerises and ruptures the bilayer. Reason: Core location, established by cryo-EM and super-resolution imaging of NINJ1 assemblies in the membranes of dying cells and consistent across the IBA, the UniProt mapping and four independent IDAs. Supporting Evidence: PMID:37198476 Super-resolution microscopy reveals that NINJ1 clusters into structurally diverse assemblies in the membranes of dying cells, in particular large, filamentous assemblies with branched morphology. |
| GO:0005886 plasma membrane | IDA PMID:38614101 NINJ1 mediates plasma membrane rupture by cutting and releas... | ACCEPT | Summary: NINJ1 is a multi-pass integral protein of the plasma membrane, and this is where it oligomerises and ruptures the bilayer. Reason: Core location, established by cryo-EM and super-resolution imaging of NINJ1 assemblies in the membranes of dying cells and consistent across the IBA, the UniProt mapping and four independent IDAs. Supporting Evidence: PMID:37198476 Super-resolution microscopy reveals that NINJ1 clusters into structurally diverse assemblies in the membranes of dying cells, in particular large, filamentous assemblies with branched morphology. |
| GO:0019835 cytolysis | IDA PMID:33472215 NINJ1 mediates plasma membrane rupture during lytic cell dea... | ACCEPT | Summary: NINJ1 execution of plasma membrane rupture, that is, the loss of membrane integrity and release of cytoplasmic contents at the end of lytic cell death. Reason: The core biological process and the closest term GO currently offers to plasma membrane rupture. Directly demonstrated: Ninj1-null macrophages die but fail to release LDH and HMGB1 and remain as persistent ballooned corpses. Supporting Evidence: PMID:33472215 Ninj1-/- macrophages exhibited impaired PMR in response to diverse inducers of pyroptotic, necrotic and apoptotic cell death, and were unable to release numerous intracellular proteins including HMGB1 (a known DAMP) and LDH (a standard measure of PMR). |
| GO:0019835 cytolysis | IDA PMID:38614101 NINJ1 mediates plasma membrane rupture by cutting and releas... | ACCEPT | Summary: NINJ1 execution of plasma membrane rupture, that is, the loss of membrane integrity and release of cytoplasmic contents at the end of lytic cell death. Reason: The core biological process and the closest term GO currently offers to plasma membrane rupture. Directly demonstrated: Ninj1-null macrophages die but fail to release LDH and HMGB1 and remain as persistent ballooned corpses. Supporting Evidence: PMID:33472215 Ninj1-/- macrophages exhibited impaired PMR in response to diverse inducers of pyroptotic, necrotic and apoptotic cell death, and were unable to release numerous intracellular proteins including HMGB1 (a known DAMP) and LDH (a standard measure of PMR). |
| GO:0051260 protein homooligomerization | IDA PMID:38396301 NINJ1 induces plasma membrane rupture and release of damage-... | ACCEPT | Summary: Activation converts NINJ1 from an autoinhibited dimer into long branched filaments and rings in the plasma membrane; this self-association is the activation step itself. Reason: Core. Two cryo-EM structures resolve the filament, an antibody that blocks oligomerisation blocks rupture, and glycine - the classical cytoprotectant - acts by preventing NINJ1 clustering. Oligomerisation and membrane destabilisation are not separable events for this protein. Supporting Evidence: PMID:36468682 Next, we show that glycine prevents NINJ1 clustering by either direct or indirect mechanisms. PMID:37198476 A cryo-electron microscopy structure of NINJ1 filaments shows a tightly packed fence-like array of transmembrane Ξ±-helices. |
| GO:0051260 protein homooligomerization | IDA PMID:38614101 NINJ1 mediates plasma membrane rupture by cutting and releas... | ACCEPT | Summary: Activation converts NINJ1 from an autoinhibited dimer into long branched filaments and rings in the plasma membrane; this self-association is the activation step itself. Reason: Core. Two cryo-EM structures resolve the filament, an antibody that blocks oligomerisation blocks rupture, and glycine - the classical cytoprotectant - acts by preventing NINJ1 clustering. Oligomerisation and membrane destabilisation are not separable events for this protein. Supporting Evidence: PMID:36468682 Next, we show that glycine prevents NINJ1 clustering by either direct or indirect mechanisms. PMID:37198476 A cryo-electron microscopy structure of NINJ1 filaments shows a tightly packed fence-like array of transmembrane Ξ±-helices. |
| GO:0071474 cellular hyperosmotic response | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Rupture of osmotically swollen cells, transferred from mouse Ninj1. Reason: Plausible and mechanistically continuous with the core activity - NINJ1 finishes cells that have swollen - but supported for human NINJ1 only by orthology to a mouse annotation that UniProt itself records as 'By similarity'. Retained, not treated as core. |
| GO:0097300 programmed necrotic cell death | IDA PMID:33472215 NINJ1 mediates plasma membrane rupture during lytic cell dea... | ACCEPT | Summary: NINJ1 also executes membrane rupture in necrotic forms of programmed death. Reason: Same single activity acting in another death program; supported by the founding forward-genetic study, which tested pyroptotic, necrotic and apoptotic inducers in parallel. Supporting Evidence: PMID:33472215 Ninj1-/- macrophages exhibited impaired PMR in response to diverse inducers of pyroptotic, necrotic and apoptotic cell death, and were unable to release numerous intracellular proteins including HMGB1 (a known DAMP) and LDH (a standard measure of PMR). |
| GO:0097707 ferroptosis | ISS GO_REF:0000024 | ACCEPT | Summary: NINJ1 requirement for the rupture step of ferroptosis, transferred from mouse Ninj1. Reason: The orthology transfer rests on a well-controlled study showing that NINJ1 oligomerises during ferroptosis and that Ninj1 deficiency protects against ferroptosis-associated rupture while leaving lipid peroxidation, calcium influx and swelling intact - that is, NINJ1 acts at the same terminal step here as elsewhere, not in ferroptosis initiation. Supporting Evidence: PMID:38396301 We report that NINJ1 oligomerizes during ferroptosis, and that Ninj1-deficiency protects macrophages and fibroblasts from ferroptosis-associated PMR. PMID:38396301 Mechanistically, we find that NINJ1 is dispensable for the initial steps of ferroptosis, such as lipid peroxidation, channel-mediated calcium influx, and cell swelling. |
| GO:0140912 membrane destabilizing activity | IDA PMID:38614101 NINJ1 mediates plasma membrane rupture by cutting and releas... | ACCEPT | Summary: NINJ1 binds the plasma membrane and destroys its integrity: it polymerises into amphipathic filaments whose hydrophilic face caps a membrane edge, so membrane material is shed rather than merely perforated. Reason: The core molecular function, and the single best-evidenced thing NINJ1 does. Supported by six independent IDAs (forward-genetic screen, antibody blockade, two cryo-EM structures, reconstitution with recombinant protein) plus the phylogenetic inference. The mechanistic detail is still argued - Degen et al. and Dai and colleagues describe filaments that cap or solubilise membrane fragments, whereas Sahoo et al. also see less frequent pore-like assemblies - but every model is an instance of this term, so the disagreement is about mechanism, not about whether the activity exists. Supporting Evidence: PMID:37198476 Our data thus suggest that, during lytic cell death, the extracellular Ξ±-helices of NINJ1 insert into the plasma membrane to polymerize NINJ1 monomers into amphipathic filaments that rupture the plasma membrane. PMID:38614101 This structural observation suggests that NINJ1 can form membrane disks, consistent with membrane fragmentation by recombinant NINJ1. PMID:39667936 This structural feature and other evidence point to a PMR mechanism by which NINJ1 filaments wrap around and solubilize membrane fragments and, less frequently, form pores in the plasma membrane. PMID:33472215 Mechanistically, NINJ1 used an evolutionarily conserved extracellular domain for oligomerization and subsequent PMR. |
| GO:0141201 pyroptotic cell death | IDA PMID:36468682 Glycine inhibits NINJ1 membrane clustering to suppress plasm... | ACCEPT | Summary: NINJ1 carries out the terminal membrane-rupture step of pyroptosis, downstream of gasdermin pore formation. Reason: Best-evidenced of the cell-death contexts in which NINJ1 acts, with four human IDAs and the phylogenetic inference. Worth stating plainly that NINJ1 is not pyroptosis-specific: it performs the same act after apoptosis, in necroptosis and in ferroptosis, and in glucose-starved macrophages it ruptures membranes with no known death program engaged at all. The term is accepted as a genuine context of the one core activity, not as evidence of pathway specificity. Supporting Evidence: PMID:37196676 Here we describe an anti-NINJ1 monoclonal antibody that specifically targets mouse NINJ1 and blocks oligomerization of NINJ1, preventing PMR. PMID:42270656 In glucose-starved macrophages, NINJ1 ruptures membranes independently of known cell death programs. |
| GO:0141201 pyroptotic cell death | IDA PMID:37196676 Inhibiting membrane rupture with NINJ1 antibodies limits tis... | ACCEPT | Summary: NINJ1 carries out the terminal membrane-rupture step of pyroptosis, downstream of gasdermin pore formation. Reason: Best-evidenced of the cell-death contexts in which NINJ1 acts, with four human IDAs and the phylogenetic inference. Worth stating plainly that NINJ1 is not pyroptosis-specific: it performs the same act after apoptosis, in necroptosis and in ferroptosis, and in glucose-starved macrophages it ruptures membranes with no known death program engaged at all. The term is accepted as a genuine context of the one core activity, not as evidence of pathway specificity. Supporting Evidence: PMID:37196676 Here we describe an anti-NINJ1 monoclonal antibody that specifically targets mouse NINJ1 and blocks oligomerization of NINJ1, preventing PMR. PMID:42270656 In glucose-starved macrophages, NINJ1 ruptures membranes independently of known cell death programs. |
| GO:0141201 pyroptotic cell death | IDA PMID:37198476 Structural basis of NINJ1-mediated plasma membrane rupture i... | ACCEPT | Summary: NINJ1 carries out the terminal membrane-rupture step of pyroptosis, downstream of gasdermin pore formation. Reason: Best-evidenced of the cell-death contexts in which NINJ1 acts, with four human IDAs and the phylogenetic inference. Worth stating plainly that NINJ1 is not pyroptosis-specific: it performs the same act after apoptosis, in necroptosis and in ferroptosis, and in glucose-starved macrophages it ruptures membranes with no known death program engaged at all. The term is accepted as a genuine context of the one core activity, not as evidence of pathway specificity. Supporting Evidence: PMID:37196676 Here we describe an anti-NINJ1 monoclonal antibody that specifically targets mouse NINJ1 and blocks oligomerization of NINJ1, preventing PMR. PMID:42270656 In glucose-starved macrophages, NINJ1 ruptures membranes independently of known cell death programs. |
| GO:0141201 pyroptotic cell death | IDA PMID:38614101 NINJ1 mediates plasma membrane rupture by cutting and releas... | ACCEPT | Summary: NINJ1 carries out the terminal membrane-rupture step of pyroptosis, downstream of gasdermin pore formation. Reason: Best-evidenced of the cell-death contexts in which NINJ1 acts, with four human IDAs and the phylogenetic inference. Worth stating plainly that NINJ1 is not pyroptosis-specific: it performs the same act after apoptosis, in necroptosis and in ferroptosis, and in glucose-starved macrophages it ruptures membranes with no known death program engaged at all. The term is accepted as a genuine context of the one core activity, not as evidence of pathway specificity. Supporting Evidence: PMID:37196676 Here we describe an anti-NINJ1 monoclonal antibody that specifically targets mouse NINJ1 and blocks oligomerization of NINJ1, preventing PMR. PMID:42270656 In glucose-starved macrophages, NINJ1 ruptures membranes independently of known cell death programs. |
| GO:0005886 plasma membrane | IDA PMID:37198476 Structural basis of NINJ1-mediated plasma membrane rupture i... | ACCEPT | Summary: NINJ1 is a multi-pass integral protein of the plasma membrane, and this is where it oligomerises and ruptures the bilayer. Reason: Core location, established by cryo-EM and super-resolution imaging of NINJ1 assemblies in the membranes of dying cells and consistent across the IBA, the UniProt mapping and four independent IDAs. Supporting Evidence: PMID:37198476 Super-resolution microscopy reveals that NINJ1 clusters into structurally diverse assemblies in the membranes of dying cells, in particular large, filamentous assemblies with branched morphology. |
| GO:0051260 protein homooligomerization | IDA PMID:37196676 Inhibiting membrane rupture with NINJ1 antibodies limits tis... | ACCEPT | Summary: Activation converts NINJ1 from an autoinhibited dimer into long branched filaments and rings in the plasma membrane; this self-association is the activation step itself. Reason: Core. Two cryo-EM structures resolve the filament, an antibody that blocks oligomerisation blocks rupture, and glycine - the classical cytoprotectant - acts by preventing NINJ1 clustering. Oligomerisation and membrane destabilisation are not separable events for this protein. Supporting Evidence: PMID:36468682 Next, we show that glycine prevents NINJ1 clustering by either direct or indirect mechanisms. PMID:37198476 A cryo-electron microscopy structure of NINJ1 filaments shows a tightly packed fence-like array of transmembrane Ξ±-helices. |
| GO:0051260 protein homooligomerization | IDA PMID:37198476 Structural basis of NINJ1-mediated plasma membrane rupture i... | ACCEPT | Summary: Activation converts NINJ1 from an autoinhibited dimer into long branched filaments and rings in the plasma membrane; this self-association is the activation step itself. Reason: Core. Two cryo-EM structures resolve the filament, an antibody that blocks oligomerisation blocks rupture, and glycine - the classical cytoprotectant - acts by preventing NINJ1 clustering. Oligomerisation and membrane destabilisation are not separable events for this protein. Supporting Evidence: PMID:36468682 Next, we show that glycine prevents NINJ1 clustering by either direct or indirect mechanisms. PMID:37198476 A cryo-electron microscopy structure of NINJ1 filaments shows a tightly packed fence-like array of transmembrane Ξ±-helices. |
| GO:0140912 membrane destabilizing activity | IDA PMID:36468682 Glycine inhibits NINJ1 membrane clustering to suppress plasm... | ACCEPT | Summary: NINJ1 binds the plasma membrane and destroys its integrity: it polymerises into amphipathic filaments whose hydrophilic face caps a membrane edge, so membrane material is shed rather than merely perforated. Reason: The core molecular function, and the single best-evidenced thing NINJ1 does. Supported by six independent IDAs (forward-genetic screen, antibody blockade, two cryo-EM structures, reconstitution with recombinant protein) plus the phylogenetic inference. The mechanistic detail is still argued - Degen et al. and Dai and colleagues describe filaments that cap or solubilise membrane fragments, whereas Sahoo et al. also see less frequent pore-like assemblies - but every model is an instance of this term, so the disagreement is about mechanism, not about whether the activity exists. Supporting Evidence: PMID:37198476 Our data thus suggest that, during lytic cell death, the extracellular Ξ±-helices of NINJ1 insert into the plasma membrane to polymerize NINJ1 monomers into amphipathic filaments that rupture the plasma membrane. PMID:38614101 This structural observation suggests that NINJ1 can form membrane disks, consistent with membrane fragmentation by recombinant NINJ1. PMID:39667936 This structural feature and other evidence point to a PMR mechanism by which NINJ1 filaments wrap around and solubilize membrane fragments and, less frequently, form pores in the plasma membrane. PMID:33472215 Mechanistically, NINJ1 used an evolutionarily conserved extracellular domain for oligomerization and subsequent PMR. |
| GO:0140912 membrane destabilizing activity | IDA PMID:37196676 Inhibiting membrane rupture with NINJ1 antibodies limits tis... | ACCEPT | Summary: NINJ1 binds the plasma membrane and destroys its integrity: it polymerises into amphipathic filaments whose hydrophilic face caps a membrane edge, so membrane material is shed rather than merely perforated. Reason: The core molecular function, and the single best-evidenced thing NINJ1 does. Supported by six independent IDAs (forward-genetic screen, antibody blockade, two cryo-EM structures, reconstitution with recombinant protein) plus the phylogenetic inference. The mechanistic detail is still argued - Degen et al. and Dai and colleagues describe filaments that cap or solubilise membrane fragments, whereas Sahoo et al. also see less frequent pore-like assemblies - but every model is an instance of this term, so the disagreement is about mechanism, not about whether the activity exists. Supporting Evidence: PMID:37198476 Our data thus suggest that, during lytic cell death, the extracellular Ξ±-helices of NINJ1 insert into the plasma membrane to polymerize NINJ1 monomers into amphipathic filaments that rupture the plasma membrane. PMID:38614101 This structural observation suggests that NINJ1 can form membrane disks, consistent with membrane fragmentation by recombinant NINJ1. PMID:39667936 This structural feature and other evidence point to a PMR mechanism by which NINJ1 filaments wrap around and solubilize membrane fragments and, less frequently, form pores in the plasma membrane. PMID:33472215 Mechanistically, NINJ1 used an evolutionarily conserved extracellular domain for oligomerization and subsequent PMR. |
| GO:0140912 membrane destabilizing activity | IDA PMID:37198476 Structural basis of NINJ1-mediated plasma membrane rupture i... | ACCEPT | Summary: NINJ1 binds the plasma membrane and destroys its integrity: it polymerises into amphipathic filaments whose hydrophilic face caps a membrane edge, so membrane material is shed rather than merely perforated. Reason: The core molecular function, and the single best-evidenced thing NINJ1 does. Supported by six independent IDAs (forward-genetic screen, antibody blockade, two cryo-EM structures, reconstitution with recombinant protein) plus the phylogenetic inference. The mechanistic detail is still argued - Degen et al. and Dai and colleagues describe filaments that cap or solubilise membrane fragments, whereas Sahoo et al. also see less frequent pore-like assemblies - but every model is an instance of this term, so the disagreement is about mechanism, not about whether the activity exists. Supporting Evidence: PMID:37198476 Our data thus suggest that, during lytic cell death, the extracellular Ξ±-helices of NINJ1 insert into the plasma membrane to polymerize NINJ1 monomers into amphipathic filaments that rupture the plasma membrane. PMID:38614101 This structural observation suggests that NINJ1 can form membrane disks, consistent with membrane fragmentation by recombinant NINJ1. PMID:39667936 This structural feature and other evidence point to a PMR mechanism by which NINJ1 filaments wrap around and solubilize membrane fragments and, less frequently, form pores in the plasma membrane. PMID:33472215 Mechanistically, NINJ1 used an evolutionarily conserved extracellular domain for oligomerization and subsequent PMR. |
| GO:0140912 membrane destabilizing activity | IDA PMID:33472215 NINJ1 mediates plasma membrane rupture during lytic cell dea... | ACCEPT | Summary: NINJ1 binds the plasma membrane and destroys its integrity: it polymerises into amphipathic filaments whose hydrophilic face caps a membrane edge, so membrane material is shed rather than merely perforated. Reason: The core molecular function, and the single best-evidenced thing NINJ1 does. Supported by six independent IDAs (forward-genetic screen, antibody blockade, two cryo-EM structures, reconstitution with recombinant protein) plus the phylogenetic inference. The mechanistic detail is still argued - Degen et al. and Dai and colleagues describe filaments that cap or solubilise membrane fragments, whereas Sahoo et al. also see less frequent pore-like assemblies - but every model is an instance of this term, so the disagreement is about mechanism, not about whether the activity exists. Supporting Evidence: PMID:37198476 Our data thus suggest that, during lytic cell death, the extracellular Ξ±-helices of NINJ1 insert into the plasma membrane to polymerize NINJ1 monomers into amphipathic filaments that rupture the plasma membrane. PMID:38614101 This structural observation suggests that NINJ1 can form membrane disks, consistent with membrane fragmentation by recombinant NINJ1. PMID:39667936 This structural feature and other evidence point to a PMR mechanism by which NINJ1 filaments wrap around and solubilize membrane fragments and, less frequently, form pores in the plasma membrane. PMID:33472215 Mechanistically, NINJ1 used an evolutionarily conserved extracellular domain for oligomerization and subsequent PMR. |
| GO:0001530 lipopolysaccharide binding | IDA PMID:26677008 Ninjurin1 regulates lipopolysaccharide-induced inflammation ... | KEEP AS NON CORE | Summary: Direct binding of biotinylated LPS to NINJ1, mapped to residues 81-100. Reason: A real pull-down result, but a single-laboratory finding in transfected HEK293T lysates that has not been independently reproduced, has no structural counterpart in the several cryo-EM structures of NINJ1, and is not part of the rupture mechanism. The residues implicated (81-100) lie outside the alpha1/alpha2 amphipathic region that carries the characterised activity. Kept because it is experimental, demoted because it is unreplicated and mechanically unconnected to the rest of NINJ1 biology. Supporting Evidence: PMID:26677008 Notably, pull-down assays using lysates from HEK293T cells transfected with human or mouse Ninjurin1 and biotinylated LPS (LPS-biotin) showed that LPS directly bound Ninjurin1. |
| GO:0002232 leukocyte chemotaxis involved in inflammatory response | IDA PMID:22162058 Role of Ninjurin-1 in the migration of myeloid cells to cent... | KEEP AS NON CORE | Summary: NINJ1 blockade abrogates monocyte adhesion to and migration across blood-brain-barrier endothelial cells and reduces myeloid infiltration in EAE. Reason: A well-controlled experimental result, but a tissue- and cell-type-specific consequence of the adhesion activity rather than a function NINJ1 protein performs in its own right. Supporting Evidence: PMID:22162058 Moreover, Ninjurin-1 neutralization specifically abrogated the adhesion and migration of human monocytes across BBB-ECs, without affecting lymphocyte recruitment. |
| GO:0007155 cell adhesion | IDA PMID:33028854 Ninjurin 1 dodecamer peptide containing the N-terminal adhes... | ACCEPT | Summary: Homophilic adhesion between cells expressing NINJ1. Reason: The biological-process counterpart of the retained cell-cell adhesion mediator activity, supported by aggregation assays in the founding work, by the N-NAM peptide study and by the phylogenetic inference across the ninjurin family including the Drosophila members. Supporting Evidence: PMID:9261151 Aggregation assays were used to demonstrate that ninjurin-mediated adhesion requires divalent cations and is an energy-dependent process. |
| GO:0034113 heterotypic cell-cell adhesion | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Adhesion between cells of different types, for example monocytes to endothelium, transferred from mouse Ninj1. Reason: Consistent with the demonstrated role of NINJ1 in monocyte transendothelial migration, but this human annotation is orthology-based only, and the underlying adhesion is homophilic NINJ1-NINJ1 binding between two cell types rather than a distinct heterotypic activity. |
| GO:0034145 positive regulation of toll-like receptor 4 signaling pathway | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Positive regulation of TLR4 signalling, transferred from mouse Ninj1. Reason: Rests on the same single-laboratory LPS-binding study as the lipopolysaccharide binding annotation, propagated through the mouse ortholog. Kept for consistency with that annotation and demoted for the same reason: unreplicated and unconnected to the structurally characterised activity. |
| GO:0042692 muscle cell differentiation | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Muscle cell differentiation, transferred from mouse Ninj1. Reason: UniProt records the striated-muscle role for NINJ1 only as 'By similarity'; there is no direct human evidence and no proposed mechanism. Retained as a low-confidence peripheral annotation. |
| GO:0045766 positive regulation of angiogenesis | IDA PMID:33028854 Ninjurin 1 dodecamer peptide containing the N-terminal adhes... | KEEP AS NON CORE | Summary: An N-terminal adhesion-motif peptide that antagonises endogenous NINJ1 promotes endothelial proliferation, migration and tube formation, implying that NINJ1 itself restrains these. Reason: Downstream tissue-level physiology, and note the direction of the underlying experiment: the pro-angiogenic effect was produced by suppressing endogenous NINJ1 with the N-NAM peptide, so 'positive regulation of angiogenesis' is not the most transparent reading of that result. Retained as peripheral rather than corrected, because this is an experimental annotation whose full text was read by the curator and the rat orthology transfer agrees with it. Supporting Evidence: PMID:33028854 We found that N-NAM promotes proliferation, migration, and tube formation of HUVECs and demonstrate that the suppression of endogenous Ninj1 is responsible for the N-NAM-mediated pro-angiogenic effects. |
| GO:0050729 positive regulation of inflammatory response | IDA PMID:26677008 Ninjurin1 regulates lipopolysaccharide-induced inflammation ... | ACCEPT | Summary: NINJ1 amplifies inflammation, both by releasing DAMPs when it ruptures the membrane and, per the LPS study, by supporting LPS-driven nitric oxide and TNF-alpha output. Reason: Core downstream process: the biological point of NINJ1-mediated rupture is that it converts a dying cell into a source of pro-inflammatory DAMPs, which is why NINJ1 blockade is anti-inflammatory in liver injury models. Supporting Evidence: PMID:37196676 Accordingly, serum levels of lactate dehydrogenase, the liver enzymes alanine aminotransaminase and aspartate aminotransferase, and the DAMPs interleukin 18 and HMGB1 were reduced. |
| GO:0051260 protein homooligomerization | IDA PMID:33472215 NINJ1 mediates plasma membrane rupture during lytic cell dea... | ACCEPT | Summary: Activation converts NINJ1 from an autoinhibited dimer into long branched filaments and rings in the plasma membrane; this self-association is the activation step itself. Reason: Core. Two cryo-EM structures resolve the filament, an antibody that blocks oligomerisation blocks rupture, and glycine - the classical cytoprotectant - acts by preventing NINJ1 clustering. Oligomerisation and membrane destabilisation are not separable events for this protein. Supporting Evidence: PMID:36468682 Next, we show that glycine prevents NINJ1 clustering by either direct or indirect mechanisms. PMID:37198476 A cryo-electron microscopy structure of NINJ1 filaments shows a tightly packed fence-like array of transmembrane Ξ±-helices. |
| GO:0098631 cell adhesion mediator activity | IDA PMID:33028854 Ninjurin 1 dodecamer peptide containing the N-terminal adhes... | MODIFY | Summary: Parent adhesion-mediator term, from the N-NAM peptide study and by orthology from rat Ninj1. Reason: Correct in kind but under-specific. The demonstrated activity is homophilic NINJ1-NINJ1 binding between apposed cells, which the child term states exactly; the parent would also cover adhesion to an extracellular substrate, which has not been shown. Proposed replacements: cell-cell adhesion mediator activity Supporting Evidence: PMID:33028854 Importantly, a pull-down assay revealed a direct binding between exogenously delivered N-NAM and endogenous Ninj1 and it is N-terminal adhesion motif dependent. |
| GO:0007155 cell adhesion | TAS PMID:9261151 Mechanism of homophilic binding mediated by ninjurin, a nove... | ACCEPT | Summary: Homophilic adhesion between cells expressing NINJ1. Reason: The biological-process counterpart of the retained cell-cell adhesion mediator activity, supported by aggregation assays in the founding work, by the N-NAM peptide study and by the phylogenetic inference across the ninjurin family including the Drosophila members. Supporting Evidence: PMID:9261151 Aggregation assays were used to demonstrate that ninjurin-mediated adhesion requires divalent cations and is an energy-dependent process. |
| GO:0007399 nervous system development | TAS PMID:9261151 Mechanism of homophilic binding mediated by ninjurin, a nove... | KEEP AS NON CORE | Summary: Nervous-system development, from the founding characterisation of ninjurin as a nerve injury-induced adhesion molecule that promotes neurite outgrowth from DRG neurons. Reason: A developmental context in which the adhesion activity is used, not a core function. Note that the same paper reports ninjurin is widely expressed in adult and embryonic tissues, mostly epithelial, so the neural framing reflects how the gene was found rather than where it principally acts. Supporting Evidence: PMID:9261151 We have now found that ninjurin is widely expressed in both adult and embryonic tissues, primarily in those of epithelial origin. |
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Download this section (compressed HTML)Q: Do NINJ1 filaments rupture the membrane by excising and shedding membrane disks, or by enlarging into very large pores? Wu and colleagues and Dai and colleagues both resolve filaments that can cap a membrane edge, but the first describes released ring-encircled membrane disks and the second also observes less frequent pores. Are these two outcomes of one polymerisation reaction that differ by filament curvature and membrane tension, or genuinely different mechanisms?
Q: Is the homophilic adhesion activity of the N-terminal motif mechanistically the same event as the membrane-inserting polymerisation, using the same extracellular helices in trans rather than in cis - and if so, what switches between them?
Q: Which NINJ1 activity supports haematopoietic stem and progenitor cell amplification? PMR-deficient Ninj1 mutants retain the function and it requires canonical WNT signalling, but neither the adhesion motif nor a NINJ1-WNT physical link has been tested.
Q: What is the proximal trigger for NINJ1 activation? It fires downstream of gasdermins, MLKL, apoptosis, lipid peroxidation, osmotic swelling and glucose starvation alike, which suggests it reads a shared membrane property - tension, lipid packing or loss of a lipid asymmetry - rather than a pathway-specific signal.
Q: Is the secreted, cleaved form of NINJ1 a physiologically significant species in humans, which protease generates it, and does it act through the same adhesion motif?
Experiment: Reconstitute purified human NINJ1 into giant unilamellar vesicles held at defined, tunable membrane tension with a micropipette aspiration setup, and image filament growth and membrane fate by high-speed AFM and cryo-ET in parallel. Score the frequency of closed rings that release disks versus open arcs that leave persistent large apertures as a function of tension, cholesterol content and NINJ1 surface density.
Hypothesis: The membrane-disk and large-pore outcomes are the same polymerisation reaction resolved under different membrane tension.
Experiment: Build a panel of NINJ1 point mutants that separately disrupt the Pro26-Asn37 adhesive determinant and the hydrophilic faces of alpha1/alpha2, knock them into the endogenous locus of human macrophages and endothelial cells, and assay each line for cell aggregation, monocyte transendothelial migration, and LDH/HMGB1 release after nigericin, TNF plus SMAC mimetic, and RSL3. A mutant that is adhesion-null but rupture-competent, or the converse, would settle whether these are one activity or two.
Hypothesis: The N-terminal adhesion motif and the alpha1/alpha2 rupture surface are separable, so adhesion and PMR can be uncoupled by mutation.
Experiment: In human CD34+ HSPCs, compare NINJ1 knockout with knock-in of the PMR-deficient and the adhesion-motif-deficient alleles, measuring expansion, WNT reporter activity and beta-catenin stabilisation. Complement with a competitive N-NAM peptide and with proximity labelling from NINJ1 in HSPCs to ask whether WNT pathway components are in its neighbourhood.
Hypothesis: NINJ1 supports haematopoietic stem and progenitor cell expansion through its adhesion motif rather than through membrane rupture.
Experiment: Use a NINJ1 conformational FRET reporter to time the dimer-to-filament transition in single live cells while independently imaging membrane tension (Flipper-TR), phospholipid scrambling and cytosolic ion changes across pyroptotic, ferroptotic, necroptotic and glucose-starvation stimuli. Then test sufficiency by imposing each candidate change in isolation - mechanical stretch, scramblase activation, controlled K+ efflux - in cells with no death program engaged.
Hypothesis: NINJ1 activation is triggered by a shared physical change in the plasma membrane rather than by any pathway-specific protein signal.
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