Humanin (HN) is a 24-residue peptide translated from a short open reading frame inside MT-RNR2, the mitochondrial 16S ribosomal RNA gene, and is the founding member of the mitochondrial-derived peptide family that also includes MOTS-c and the small humanin-like peptides SHLP1-6. It behaves as a secreted, cytoprotective signalling peptide: it is released from cells, circulates in plasma at levels that decline with age, and engages cell-surface receptors - the G protein-coupled formyl peptide receptors FPR2/FPRL1 and FPR3/FPRL2, and a gp130-family cytokine receptor complex containing CNTFR and WSX-1/IL27RA - triggering JAK/STAT3 signalling that protects neurons, retinal pigment epithelium, endothelium and pancreatic beta-cells from apoptotic insults. Humanin also has an intracellular arm: in the cytosol it binds the BCL-2-family proteins BAX, BID and the BimEL isoform of BIM, co-assembling with them into fibers that sequester them and prevent mitochondrial outer-membrane permeabilization, and it binds IGFBP3, blocking IGFBP3-dependent cell death and competing with importin-beta1 for IGFBP3. It binds amyloid-beta oligomers directly and delays amyloid fibril formation. Humanin homodimerizes through Ser7, and the determinants of its secretion and of its neuroprotective activity have been mapped residue by residue. It is detected in testis, sperm (concentrated in the flagellar midpiece), retina, heart, skeletal muscle, kidney and liver, and intracellularly in cytoplasm and mitochondria. Two features of the peptide remain unresolved: whether the active peptide is translated from the mitochondrial transcript (which, read with the mitochondrial genetic code, would yield a 21-residue product lacking the three C-terminal residues) or from one of the thirteen nuclear MT-RNR2-like loci that derive from mitochondrial DNA inserted into the nuclear genome; and how a peptide encoded in the mitochondrial genome reaches the extracellular space. Most functional data come from synthetic peptide applied exogenously, often as the more potent S14G analogue.
Definition: Binding to an insulin-like growth factor binding protein (IGFBP), a secreted carrier protein of the IGFBP family.
Justification: Humanin binds IGFBP3 with high affinity and specificity, the interaction has been mapped to the IGFBP3 heparin-binding segment (residues 215-232), is abolished by the humanin F6A substitution, and has defined consequences: humanin blocks IGFBP3-induced cell death and competes with importin-beta1 for IGFBP3, preventing its nuclear import. Three independent GOA rows record this interaction as bare GO:0005515 protein binding, which conveys none of it. No existing GO term fits: GO:0005520 insulin-like growth factor binding and its children describe binding to IGF itself, not to the carrier, and GO:0019838 growth factor binding is wrong for the same reason. The term would also serve other IGFBP-binding proteins (for example the humanin-competitive hyaluronan-CD44 system described in PMID:30184438) rather than being gene-specific.
Parent term: protein binding
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0001664 G protein-coupled receptor binding | IPI PMID:15465011 N-Formylated humanin activates both formyl peptide receptor-... | ACCEPT | Summary: Direct binding of humanin to FPR2/FPRL1 (UniProtKB:P25089), shown by suppression of forskolin-induced cAMP production in CHO cells expressing the human receptor, by radioligand competition on membranes, and by chemotaxis of the transfected cells; EC50 for HN at FPRL1 is 3.5 nM. Reason: Well-supported and informative: the formyl peptide receptors are the best-characterised cell-surface receptors for humanin, and this row names the specific receptor bound. This is one of humanin's core molecular functions. Supporting Evidence: PMID:15465011 We have discovered that humanin (HN) acts as a ligand for formyl peptide receptor-like 1 (FPRL1) and 2 (FPRL2) PMID:15465011 We demonstrated by binding experiments using [(125)I]-W peptide that HN and fHN directly interacted with hFPRL1 on the membrane |
| GO:0001664 G protein-coupled receptor binding | IPI PMID:15465011 N-Formylated humanin activates both formyl peptide receptor-... | ACCEPT | Summary: Direct binding of humanin to FPR3/FPRL2 (UniProtKB:P25090), shown by suppression of forskolin-induced cAMP production in CHO cells expressing the human receptor, by radioligand competition on membranes, and by chemotaxis of the transfected cells; EC50 for HN at FPRL1 is 3.5 nM. Reason: Well-supported and informative: the formyl peptide receptors are the best-characterised cell-surface receptors for humanin, and this row names the specific receptor bound. This is one of humanin's core molecular functions. Supporting Evidence: PMID:15465011 We have discovered that humanin (HN) acts as a ligand for formyl peptide receptor-like 1 (FPRL1) and 2 (FPRL2) PMID:15465011 We demonstrated by binding experiments using [(125)I]-W peptide that HN and fHN directly interacted with hFPRL1 on the membrane |
| GO:0005102 signaling receptor binding | TAS PMID:15661737 Humanin binds and nullifies Bid activity by blocking its act... | MODIFY | Summary: Generic parent term asserted as a traceable author statement from the BID paper, whose own data concern binding of humanin to BID/tBID rather than to a receptor; the receptor claim in that paper is a citation of the FPRL1 work. Reason: Humanin does bind signalling receptors, but this row adds nothing over the two GO:0001664 IPI rows that name FPR2 and FPR3, and the cited publication is not the source of the receptor evidence. Replacing it with the specific child term keeps the assertion and attaches it to the right level of detail. Proposed replacements: G protein-coupled receptor binding Supporting Evidence: PMID:15465011 We have discovered that humanin (HN) acts as a ligand for formyl peptide receptor-like 1 (FPRL1) and 2 (FPRL2) PMID:15661737 We show here that HN also interacts with the BH3-only Bcl-2/Bax family protein, Bid, as well as a truncated form of Bid (tBid) associated with protease-mediated activation of this proapoptotic protein |
| GO:0005515 protein binding | IPI PMID:12732850 Humanin peptide suppresses apoptosis by interfering with Bax... | MODIFY | Summary: Interaction with BAX (UniProtKB:Q07812), shown by co-immunoprecipitation and in vitro binding; humanin prevents BAX translocation from cytosol to mitochondria and blocks BAX association with isolated mitochondria. Reason: A strong, mechanistically consequential interaction, but bare protein binding is uninformative and CLAUDE.md directs it to be replaced where a better molecular function exists. BAX is a BCL-2-family BH3 protein and the humanin-binding surface work places the interaction on that family, so GO:0051434 BH3 domain binding states what was shown. Later work resolves the mechanism as co-fibrillation and sequestration of BAX. Proposed replacements: BH3 domain binding Supporting Evidence: PMID:12732850 Here we show that Bax interacts with humanin (HN), an anti-apoptotic peptide of 24 amino acids encoded in mammalian genomes. HN prevents the translocation of Bax from cytosol to mitochondria PMID:31690630 Our findings reveal for the first time a potential mechanism by which BAX can be sequestered by fibril formation, which can prevent it from initiating MOMP and committing the cell to apoptosis. |
| GO:0005515 protein binding | IPI PMID:14561895 Interaction between the Alzheimer's survival peptide humanin... | MODIFY | Summary: Founding IPI for the interaction with IGFBP3 (UniProtKB:P17936): yeast two-hybrid isolation, displaceable pull-down, ligand blot, and co-immunoprecipitation from mouse testis, with the binding site mapped to the IGFBP3 heparin-binding domain and abolished by the F6A substitution. Reason: High-quality and functionally consequential (humanin blocks IGFBP3-induced cell death and competes with importin-beta1 for IGFBP3), but bare protein binding says nothing about it and GO currently has no term for binding an insulin-like-growth-factor-binding protein. GO:0005520 insulin-like growth factor binding would be wrong (humanin binds the carrier, not IGF). A new term is requested; until it exists this interaction is best left out of core_functions. Proposed replacements: insulin-like growth factor binding protein binding Supporting Evidence: PMID:14561895 By using a yeast two-hybrid screen to identify IGFBP-3-interacting proteins, we cloned humanin (HN) as an IGFBP-3-binding partner PMID:14561895 The physical interaction of HN with IGFBP-3 was determined to be of high affinity and specificity PMID:26216267 we determined that 1) humanin binds to IGFBP3 with a Kd of 5.05 |
| GO:0005515 protein binding | IPI PMID:15619032 Interaction of the spectrin-like repeats of alpha-actinin-4 ... | REMOVE | Summary: Interaction with alpha-actinin-4/ACTN4 (UniProtKB:O43707), from a bacterial two-hybrid screen of a kidney cDNA library with the actinin-4 R1-R4 rod domain as bait, confirmed by affinity assay on humanin-Sepharose and by colocalization of GFP-humanin with endogenous actinin. Reason: Bare protein binding with no informative molecular function attached and no demonstrated functional consequence: the screen was done for actinin-4 ligands, the binding maps non-specifically to all four spectrin repeats, and the colocalization uses an overexpressed GFP fusion. Per CLAUDE.md, a protein-binding row that cannot be converted to a more informative term and supports no function should be removed. Supporting Evidence: PMID:15619032 In vitro affinity assays showed specific interactions of recombinant actinin-4 R1-R4, R1, R2, R3, and R4 proteins with humanin-Sepharose PMID:15619032 Green fluorescent protein-fused humanin and endogenous actinin colocalized mainly in the perinuclear cytoplasm of HEK293 cells |
| GO:0005515 protein binding | IPI PMID:15661735 Cytoprotective peptide humanin binds and inhibits proapoptot... | MODIFY | Summary: Interaction with BIM/BCL2L11 isoform BimEL (UniProtKB:O43521-1), shown by in vitro binding, immunoprecipitation and co-immunolocalization, and isoform-selective: humanin binds BimEL but not BimL or BimS. Reason: Replacing bare protein binding with GO:0051434 BH3 domain binding, which is what the experiment establishes: BIM is a BH3-only BCL-2-family protein, humanin-binding-deficient mutants lose protection, and the protection is retained in bax-/- mitochondria. Proposed replacements: BH3 domain binding Supporting Evidence: PMID:15661735 we demonstrated that HN binds directly to the extra long isoform of Bim (BimEL) but not the long (BimL) or short (BimS) isoforms PMID:15661735 HN also protects cells against apoptosis induced by BimEL but not BimL and BimS in gene transfection studies |
| GO:0005515 protein binding | IPI PMID:15661737 Humanin binds and nullifies Bid activity by blocking its act... | MODIFY | Summary: Interaction with BID (the GOA row names the mouse ortholog UniProtKB:P70444), shown with purified BID and tBID in vitro; humanin blocks tBID-induced cytochrome c and SMAC release from isolated mitochondria and tBID-induced BAX/BAK oligomerization. Reason: Same reasoning as the BAX and BimEL rows: BID is a BH3-only BCL-2-family protein and GO:0051434 BH3 domain binding is the informative replacement for bare protein binding. The mechanism was later resolved as co-fibrillation that sequesters BID. Proposed replacements: BH3 domain binding Supporting Evidence: PMID:15661737 Synthetic HN peptide binds purified Bid and tBid in vitro and blocks tBid-induced release of cytochrome c and SMAC from isolated mitochondria PMID:33106313 Humanin selectively prevents the activation of pro-apoptotic protein BID by sequestering it into fibers. |
| GO:0005515 protein binding | IPI PMID:19386761 Humanin inhibits neuronal cell death by interacting with a c... | MODIFY | Summary: Interaction with CNTFR (UniProtKB:P26992), a subunit of the gp130-family receptor complex that mediates humanin neuroprotection: overexpression increased humanin binding to neuronal cells and siRNA knockdown reduced it, and no other tested gp130-related subunit had this effect. Reason: Bare protein binding should be replaced where a better term exists. Both partners are cytokine receptor subunits, so GO:0005126 cytokine receptor binding states what was shown and supports the receptor-ligand and STAT-signalling annotations this gene also carries. Proposed replacements: cytokine receptor binding Supporting Evidence: PMID:19386761 Overexpression of ciliary neurotrophic factor receptor alpha (CNTFR) and/or the IL-27 receptor subunit, WSX-1, but not that of any other tested gp130-related receptor subunit, up-regulated HN binding to neuronal cells, whereas siRNA-mediated knockdown of endogenous CNTFR and/or WSX-1 reduced it |
| GO:0005515 protein binding | IPI PMID:19386761 Humanin inhibits neuronal cell death by interacting with a c... | MODIFY | Summary: Interaction with IL27RA/WSX-1 (UniProtKB:Q6UWB1), a subunit of the gp130-family receptor complex that mediates humanin neuroprotection: overexpression increased humanin binding to neuronal cells and siRNA knockdown reduced it, and no other tested gp130-related subunit had this effect. Reason: Bare protein binding should be replaced where a better term exists. Both partners are cytokine receptor subunits, so GO:0005126 cytokine receptor binding states what was shown and supports the receptor-ligand and STAT-signalling annotations this gene also carries. Proposed replacements: cytokine receptor binding Supporting Evidence: PMID:19386761 Overexpression of ciliary neurotrophic factor receptor alpha (CNTFR) and/or the IL-27 receptor subunit, WSX-1, but not that of any other tested gp130-related receptor subunit, up-regulated HN binding to neuronal cells, whereas siRNA-mediated knockdown of endogenous CNTFR and/or WSX-1 reduced it |
| GO:0005515 protein binding | IPI PMID:21393573 VSTM2L is a novel secreted antagonist of the neuroprotective... | REMOVE | Summary: Interaction with VSTM2L (UniProtKB:Q96N03), a secreted protein identified as an antagonist of humanin neuroprotective activity; binding shown in yeast and mammalian cells. Reason: Bare protein binding with no informative replacement available: VSTM2L is a secreted antagonist, so the functionally meaningful assertion belongs on VSTM2L (humanin binding / neuroprotection antagonism), not as an unlabelled binding row on humanin. Nothing about humanin's own activity follows from this row. Supporting Evidence: PMID:21393573 VSTM2L interacts with HN in both yeast and mammalian cells, is secreted in cultured cells, is present in serum, and is selectively expressed in the central nervous system PMID:21393573 When tested in HN neuroprotection bioassays, VSTM2L acts as a strong antagonist of HN neuroprotective activity |
| GO:0005515 protein binding | IPI PMID:23532874 Humanin binds MPP8: mapping interaction sites of the peptide... | REMOVE | Summary: Interaction with MPP8/MPHOSPH8 (UniProtKB:Q99549), from yeast two-hybrid and confirmed by co-immunoprecipitation, with the binding site mapped to humanin residues 5-12 and MPP8 residues 431-560. Reason: Bare protein binding with no informative molecular-function replacement and no demonstrated functional consequence; the authors themselves leave the consequences for future work. A nuclear chromatin-reader partner for a secreted peptide also has no supporting cell-biological context here. Supporting Evidence: PMID:23532874 we further confirmed interaction of HN with MPP8 in co-immunoprecipitation experiments and localized an MPP8-binding site in the region between 5 and 12 aa PMID:23532874 Further studies on functional consequences of the interaction between the potential oncopetide and the oncoprotein may elucidate some aspects of the molecular mechanisms of carcinogenesis |
| GO:0005515 protein binding | IPI PMID:26216267 Humanin Peptide Binds to Insulin-Like Growth Factor-Binding ... | MODIFY | Summary: Second IPI for the humanin-IGFBP3 interaction (UniProtKB:P17936), measured by competitive ligand dot blot, co-immunoprecipitation and ELISA-based binding (Kd 5.05 uM), with humanin and HN(3-19) interfering with importin-beta1 binding to IGFBP3. Reason: Duplicates the interaction reviewed on the PMID:14561895 row; handled the same way, as a request for a term covering binding to an insulin-like-growth-factor-binding protein rather than as bare protein binding. Proposed replacements: insulin-like growth factor binding protein binding Supporting Evidence: PMID:26216267 we determined that 1) humanin binds to IGFBP3 with a Kd of 5.05 |
| GO:0005515 protein binding | IPI PMID:28282805 Humanin Specifically Interacts with Amyloid-Ξ² Oligomers and ... | MODIFY | Summary: Interaction with the amyloid-beta 42 chain of APP (UniProtKB:P05067-PRO_0000000092), shown by surface plasmon resonance against soluble Abeta42 oligomers, with humanin and HNG delaying fibril formation and reducing the final amount of fibrils. Reason: A specific informative term exists: GO:0001540 amyloid-beta binding. The binding is direct, oligomer-selective, and the basis of the negative regulation of amyloid fibril formation annotations this gene carries. Proposed replacements: amyloid-beta binding Supporting Evidence: PMID:28282805 In vitro surface plasmon resonance studies indicated that they interact with PMID:28282805 Thioflavin-T assay indicated that both HN and HNG delay the formation and reduce the final amount of |
| GO:0005515 protein binding | IPI PMID:30184438 Interaction of Insulin-Like Growth Factor-Binding Protein 3 ... | MODIFY | Summary: Third IPI for the humanin-IGFBP3 interaction (UniProtKB:P17936), from the study showing that humanin and hyaluronan compete for the same IGFBP3 215-232 segment and that humanin sequesters IGFBP3 from hyaluronan. Reason: Same interaction as the two earlier IGFBP3 rows; replaced by the same new-term request rather than retained as bare protein binding. Proposed replacements: insulin-like growth factor binding protein binding Supporting Evidence: PMID:30184438 Either HA or humanin could bind to this IGFBP-3 segment, but not simultaneously |
| GO:0005576 extracellular region | EXP PMID:19623253 Humanin: a novel central regulator of peripheral insulin act... | ACCEPT | Summary: Humanin circulates in human and rodent plasma and its levels decline with age; intracerebroventricular infusion of the peptide acts systemically. Reason: Secretion is the best-established property of humanin, is reproduced by several groups, has mapped sequence determinants, and is required for the receptor-mediated arm of its activity. The extracellular pool is where the core receptor-ligand function is exercised. Supporting Evidence: PMID:11371646 Transfected HN cDNA was transcribed to the corresponding polypeptide and then was secreted into the cultured medium. PMID:12860203 Arg substitution revealed that the two structures-Leu9-Leu11 and Pro19-Va120-were essential for the secretion of full-length HN PMID:19623253 circulating levels of HN were decreased with age in humans and mice |
| GO:0005576 extracellular region | IDA PMID:11371646 A rescue factor abolishing neuronal cell death by a wide spe... | ACCEPT | Summary: HN expressed from transfected cDNA is secreted into the culture medium, and the L9R substitution abolishes secretion. Reason: Secretion is the best-established property of humanin, is reproduced by several groups, has mapped sequence determinants, and is required for the receptor-mediated arm of its activity. The extracellular pool is where the core receptor-ligand function is exercised. Supporting Evidence: PMID:11371646 Transfected HN cDNA was transcribed to the corresponding polypeptide and then was secreted into the cultured medium. PMID:12860203 Arg substitution revealed that the two structures-Leu9-Leu11 and Pro19-Va120-were essential for the secretion of full-length HN PMID:19623253 circulating levels of HN were decreased with age in humans and mice |
| GO:0005576 extracellular region | IDA PMID:12860203 Identification of essential amino acids in Humanin, a neurop... | ACCEPT | Summary: Comprehensive mutagenesis of the secretion determinants: the Leu9-Leu11 and Pro19-Val20 segments are required for extracellular secretion of full-length HN. Reason: Secretion is the best-established property of humanin, is reproduced by several groups, has mapped sequence determinants, and is required for the receptor-mediated arm of its activity. The extracellular pool is where the core receptor-ligand function is exercised. Supporting Evidence: PMID:11371646 Transfected HN cDNA was transcribed to the corresponding polypeptide and then was secreted into the cultured medium. PMID:12860203 Arg substitution revealed that the two structures-Leu9-Leu11 and Pro19-Va120-were essential for the secretion of full-length HN PMID:19623253 circulating levels of HN were decreased with age in humans and mice |
| GO:0005576 extracellular region | IDA PMID:26990160 The Mitochondrial-Derived Peptide Humanin Protects RPE Cells... | ACCEPT | Summary: Humanin is taken up by and acts on retinal pigment epithelium cells from the medium, consistent with an extracellular pool. Reason: Secretion is the best-established property of humanin, is reproduced by several groups, has mapped sequence determinants, and is required for the receptor-mediated arm of its activity. The extracellular pool is where the core receptor-ligand function is exercised. Supporting Evidence: PMID:11371646 Transfected HN cDNA was transcribed to the corresponding polypeptide and then was secreted into the cultured medium. PMID:12860203 Arg substitution revealed that the two structures-Leu9-Leu11 and Pro19-Va120-were essential for the secretion of full-length HN PMID:19623253 circulating levels of HN were decreased with age in humans and mice |
| GO:0005576 extracellular region | IEA GO_REF:0000044 | ACCEPT | Summary: Electronic annotation from the UniProt subcellular location 'Secreted' (UniProtKB-SubCell:SL-0243). Reason: Agrees with the experimental secretion data; redundant with the IDA/EXP rows but not wrong. Supporting Evidence: PMID:11371646 Transfected HN cDNA was transcribed to the corresponding polypeptide and then was secreted into the cultured medium. |
| GO:0005576 extracellular region | TAS Reactome:R-HSA-379048 | ACCEPT | Summary: Traceable author statement from a Reactome reaction placing humanin in the extracellular compartment, where it binds the formyl peptide receptors (e.g. R-HSA-444476, FPRL2 binds humanin). Reason: Correct compartment, consistent with the experimental secretion evidence and with the FPR2/FPR3 ligand annotations. GOA carries eight such rows from the related Reactome reactions; all assert the same compartment and none is independently informative. Supporting Evidence: PMID:11371646 Transfected HN cDNA was transcribed to the corresponding polypeptide and then was secreted into the cultured medium. PMID:15465011 We have discovered that humanin (HN) acts as a ligand for formyl peptide receptor-like 1 (FPRL1) and 2 (FPRL2) |
| GO:0005576 extracellular region | TAS Reactome:R-HSA-380073 | ACCEPT | Summary: Traceable author statement from a Reactome reaction placing humanin in the extracellular compartment, where it binds the formyl peptide receptors (e.g. R-HSA-444476, FPRL2 binds humanin). Reason: Correct compartment, consistent with the experimental secretion evidence and with the FPR2/FPR3 ligand annotations. GOA carries eight such rows from the related Reactome reactions; all assert the same compartment and none is independently informative. Supporting Evidence: PMID:11371646 Transfected HN cDNA was transcribed to the corresponding polypeptide and then was secreted into the cultured medium. PMID:15465011 We have discovered that humanin (HN) acts as a ligand for formyl peptide receptor-like 1 (FPRL1) and 2 (FPRL2) |
| GO:0005576 extracellular region | TAS Reactome:R-HSA-391913 | ACCEPT | Summary: Traceable author statement from a Reactome reaction placing humanin in the extracellular compartment, where it binds the formyl peptide receptors (e.g. R-HSA-444476, FPRL2 binds humanin). Reason: Correct compartment, consistent with the experimental secretion evidence and with the FPR2/FPR3 ligand annotations. GOA carries eight such rows from the related Reactome reactions; all assert the same compartment and none is independently informative. Supporting Evidence: PMID:11371646 Transfected HN cDNA was transcribed to the corresponding polypeptide and then was secreted into the cultured medium. PMID:15465011 We have discovered that humanin (HN) acts as a ligand for formyl peptide receptor-like 1 (FPRL1) and 2 (FPRL2) |
| GO:0005576 extracellular region | TAS Reactome:R-HSA-444476 | ACCEPT | Summary: Traceable author statement from a Reactome reaction placing humanin in the extracellular compartment, where it binds the formyl peptide receptors (e.g. R-HSA-444476, FPRL2 binds humanin). Reason: Correct compartment, consistent with the experimental secretion evidence and with the FPR2/FPR3 ligand annotations. GOA carries eight such rows from the related Reactome reactions; all assert the same compartment and none is independently informative. Supporting Evidence: PMID:11371646 Transfected HN cDNA was transcribed to the corresponding polypeptide and then was secreted into the cultured medium. PMID:15465011 We have discovered that humanin (HN) acts as a ligand for formyl peptide receptor-like 1 (FPRL1) and 2 (FPRL2) |
| GO:0005576 extracellular region | TAS Reactome:R-HSA-749448 | ACCEPT | Summary: Traceable author statement from a Reactome reaction placing humanin in the extracellular compartment, where it binds the formyl peptide receptors (e.g. R-HSA-444476, FPRL2 binds humanin). Reason: Correct compartment, consistent with the experimental secretion evidence and with the FPR2/FPR3 ligand annotations. GOA carries eight such rows from the related Reactome reactions; all assert the same compartment and none is independently informative. Supporting Evidence: PMID:11371646 Transfected HN cDNA was transcribed to the corresponding polypeptide and then was secreted into the cultured medium. PMID:15465011 We have discovered that humanin (HN) acts as a ligand for formyl peptide receptor-like 1 (FPRL1) and 2 (FPRL2) |
| GO:0005576 extracellular region | TAS Reactome:R-HSA-749452 | ACCEPT | Summary: Traceable author statement from a Reactome reaction placing humanin in the extracellular compartment, where it binds the formyl peptide receptors (e.g. R-HSA-444476, FPRL2 binds humanin). Reason: Correct compartment, consistent with the experimental secretion evidence and with the FPR2/FPR3 ligand annotations. GOA carries eight such rows from the related Reactome reactions; all assert the same compartment and none is independently informative. Supporting Evidence: PMID:11371646 Transfected HN cDNA was transcribed to the corresponding polypeptide and then was secreted into the cultured medium. PMID:15465011 We have discovered that humanin (HN) acts as a ligand for formyl peptide receptor-like 1 (FPRL1) and 2 (FPRL2) |
| GO:0005576 extracellular region | TAS Reactome:R-HSA-749454 | ACCEPT | Summary: Traceable author statement from a Reactome reaction placing humanin in the extracellular compartment, where it binds the formyl peptide receptors (e.g. R-HSA-444476, FPRL2 binds humanin). Reason: Correct compartment, consistent with the experimental secretion evidence and with the FPR2/FPR3 ligand annotations. GOA carries eight such rows from the related Reactome reactions; all assert the same compartment and none is independently informative. Supporting Evidence: PMID:11371646 Transfected HN cDNA was transcribed to the corresponding polypeptide and then was secreted into the cultured medium. PMID:15465011 We have discovered that humanin (HN) acts as a ligand for formyl peptide receptor-like 1 (FPRL1) and 2 (FPRL2) |
| GO:0005576 extracellular region | TAS Reactome:R-HSA-749456 | ACCEPT | Summary: Traceable author statement from a Reactome reaction placing humanin in the extracellular compartment, where it binds the formyl peptide receptors (e.g. R-HSA-444476, FPRL2 binds humanin). Reason: Correct compartment, consistent with the experimental secretion evidence and with the FPR2/FPR3 ligand annotations. GOA carries eight such rows from the related Reactome reactions; all assert the same compartment and none is independently informative. Supporting Evidence: PMID:11371646 Transfected HN cDNA was transcribed to the corresponding polypeptide and then was secreted into the cultured medium. PMID:15465011 We have discovered that humanin (HN) acts as a ligand for formyl peptide receptor-like 1 (FPRL1) and 2 (FPRL2) |
| GO:0005634 nucleus | IDA PMID:20542501 Immunolocalization of humanin in human sperm and testis. | MARK AS OVER ANNOTATED | Summary: Immunocytochemistry and immunoelectron microscopy of human testis and ejaculated sperm report humanin immunoreactivity in the cytoplasm and nucleus of spermatocytes and spermatids and on uncoiled sperm chromatin. Reason: This is the sole evidence for a nuclear pool, it is antibody-based in a single short report with no biochemical fractionation, and no nuclear function has ever been assigned to humanin. The antibody problem is acute for this gene: thirteen nuclear MT-RNR2-like loci encode near-identical humanin-like peptides, so an anti-humanin antibody cannot attribute signal to this accession. The peptide also has no basic nuclear-targeting determinant and is a secreted ligand. Supporting Evidence: PMID:20542501 We also found HN in the cytoplasm and nucleus of spermatocytes and spermatids and in experimentally uncoiled chromatin of mature ejaculated sperm PMID:19477263 We provide bioinformatic and expression data suggesting the existence of 13 MT-RNR2-like nuclear loci predicted to maintain the open reading frames of 15 distinct full-length HN-like peptides |
| GO:0005634 nucleus | IEA GO_REF:0000044 | MARK AS OVER ANNOTATED | Summary: Electronic annotation propagating the UniProt subcellular location 'Nucleus' (UniProtKB-SubCell:SL-0191), which rests on the same single immunolocalization study. Reason: Inherits the weakness of the IDA row it derives from; no independent support. Supporting Evidence: PMID:20542501 We also found HN in the cytoplasm and nucleus of spermatocytes and spermatids and in experimentally uncoiled chromatin of mature ejaculated sperm |
| GO:0005737 cytoplasm | EXP PMID:11371646 A rescue factor abolishing neuronal cell death by a wide spe... | ACCEPT | Summary: Humanin expressed in neuronal cells is detected intracellularly as well as in the medium. Reason: A cytosolic pool is required by the best-supported intracellular mechanism: humanin holds BAX in the cytosol and prevents its translocation to mitochondria, and the BID and BimEL work is in the same compartment. The location is reported by several independent groups and by UniProt. Supporting Evidence: PMID:12732850 Here we show that Bax interacts with humanin (HN), an anti-apoptotic peptide of 24 amino acids encoded in mammalian genomes. HN prevents the translocation of Bax from cytosol to mitochondria PMID:12670303 The intracellular level of glutathione S-transferase-fused or EGFP-fused HN peptides or plain HN was drastically reduced by the coexpression of TRIM11 |
| GO:0005737 cytoplasm | EXP PMID:12670303 A tripartite motif protein TRIM11 binds and destabilizes Hum... | ACCEPT | Summary: Intracellular humanin is detectable and is destabilised by co-expression of the cytoplasmic E3 ligase TRIM11. Reason: A cytosolic pool is required by the best-supported intracellular mechanism: humanin holds BAX in the cytosol and prevents its translocation to mitochondria, and the BID and BimEL work is in the same compartment. The location is reported by several independent groups and by UniProt. Supporting Evidence: PMID:12732850 Here we show that Bax interacts with humanin (HN), an anti-apoptotic peptide of 24 amino acids encoded in mammalian genomes. HN prevents the translocation of Bax from cytosol to mitochondria PMID:12670303 The intracellular level of glutathione S-transferase-fused or EGFP-fused HN peptides or plain HN was drastically reduced by the coexpression of TRIM11 |
| GO:0005737 cytoplasm | EXP PMID:15661737 Humanin binds and nullifies Bid activity by blocking its act... | ACCEPT | Summary: Humanin acts on cytosolic BID/tBID and blocks its action on isolated mitochondria, and gene-transfected cells are protected from tBID. Reason: A cytosolic pool is required by the best-supported intracellular mechanism: humanin holds BAX in the cytosol and prevents its translocation to mitochondria, and the BID and BimEL work is in the same compartment. The location is reported by several independent groups and by UniProt. Supporting Evidence: PMID:12732850 Here we show that Bax interacts with humanin (HN), an anti-apoptotic peptide of 24 amino acids encoded in mammalian genomes. HN prevents the translocation of Bax from cytosol to mitochondria PMID:12670303 The intracellular level of glutathione S-transferase-fused or EGFP-fused HN peptides or plain HN was drastically reduced by the coexpression of TRIM11 |
| GO:0005737 cytoplasm | IDA PMID:15661735 Cytoprotective peptide humanin binds and inhibits proapoptot... | ACCEPT | Summary: Co-immunolocalization of humanin with BimEL in transfected cells. Reason: A cytosolic pool is required by the best-supported intracellular mechanism: humanin holds BAX in the cytosol and prevents its translocation to mitochondria, and the BID and BimEL work is in the same compartment. The location is reported by several independent groups and by UniProt. Supporting Evidence: PMID:12732850 Here we show that Bax interacts with humanin (HN), an anti-apoptotic peptide of 24 amino acids encoded in mammalian genomes. HN prevents the translocation of Bax from cytosol to mitochondria PMID:12670303 The intracellular level of glutathione S-transferase-fused or EGFP-fused HN peptides or plain HN was drastically reduced by the coexpression of TRIM11 |
| GO:0005737 cytoplasm | IDA PMID:20542501 Immunolocalization of humanin in human sperm and testis. | ACCEPT | Summary: Immunolocalization of humanin in the cytoplasm of spermatocytes, spermatids and sperm cytoplasmic residues. Reason: A cytosolic pool is required by the best-supported intracellular mechanism: humanin holds BAX in the cytosol and prevents its translocation to mitochondria, and the BID and BimEL work is in the same compartment. The location is reported by several independent groups and by UniProt. Supporting Evidence: PMID:12732850 Here we show that Bax interacts with humanin (HN), an anti-apoptotic peptide of 24 amino acids encoded in mammalian genomes. HN prevents the translocation of Bax from cytosol to mitochondria PMID:12670303 The intracellular level of glutathione S-transferase-fused or EGFP-fused HN peptides or plain HN was drastically reduced by the coexpression of TRIM11 |
| GO:0005737 cytoplasm | IDA PMID:26990160 The Mitochondrial-Derived Peptide Humanin Protects RPE Cells... | ACCEPT | Summary: Humanin is detected in the cytoplasm of retinal pigment epithelium cells, in addition to mitochondria. Reason: A cytosolic pool is required by the best-supported intracellular mechanism: humanin holds BAX in the cytosol and prevents its translocation to mitochondria, and the BID and BimEL work is in the same compartment. The location is reported by several independent groups and by UniProt. Supporting Evidence: PMID:12732850 Here we show that Bax interacts with humanin (HN), an anti-apoptotic peptide of 24 amino acids encoded in mammalian genomes. HN prevents the translocation of Bax from cytosol to mitochondria PMID:12670303 The intracellular level of glutathione S-transferase-fused or EGFP-fused HN peptides or plain HN was drastically reduced by the coexpression of TRIM11 |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: Electronic annotation from the UniProt subcellular location 'Cytoplasm' (UniProtKB-SubCell:SL-0086). Reason: Consistent with the experimental rows. Supporting Evidence: PMID:12732850 Here we show that Bax interacts with humanin (HN), an anti-apoptotic peptide of 24 amino acids encoded in mammalian genomes. HN prevents the translocation of Bax from cytosol to mitochondria |
| GO:0005739 mitochondrion | IDA PMID:15567815 Increased expression of humanin peptide in diffuse-type pigm... | KEEP AS NON CORE | Summary: Humanin immunoreactivity in diffuse-type pigmented villonodular synovitis localises mainly to mitochondria by double staining with HSP60 and by electron-microscopic immunohistochemistry. Reason: A mitochondrial pool is reported by three independent approaches and is unsurprising for a peptide whose ORF lies in the mitochondrial genome. Two caveats worth recording rather than acting on. The RPE result follows uptake of exogenous synthetic peptide, so it shows import rather than synthesis in the organelle, and the antibody-based results cannot distinguish this accession from the nuclear MT-RNR2-like paralogs. Neither undermines the annotation, but neither establishes that humanin is made in the matrix. ORGANELLES check (2026-11-03). HSP60 double staining, immuno-EM and co-localisation of labelled peptide with mitochondria are organelle-level readouts, the peptide has no transit peptide or transmembrane segment, and no cached study fractionates humanin into a sub-compartment, so the rows stay at the root. Moved from ACCEPT to KEEP_AS_NON_CORE because humanin's characterised activities are extracellular (receptor ligand) or cytosolic (holding BAX and tBID away from mitochondria), which is what core_functions record; the mitochondrial pool is real but not where a core activity is shown. Supporting Evidence: PMID:15567815 Double staining with anti-humanin and anti-heat shock protein 60 showed that humanin was expressed mainly in mitochondria PMID:26990160 Exogenous HN was taken up by RPE and colocalized with mitochondria. |
| GO:0005739 mitochondrion | IDA PMID:20542501 Immunolocalization of humanin in human sperm and testis. | KEEP AS NON CORE | Summary: Immunoelectron microscopy detects humanin in sperm mitochondria, concentrated in the midpiece. Reason: A mitochondrial pool is reported by three independent approaches and is unsurprising for a peptide whose ORF lies in the mitochondrial genome. Two caveats worth recording rather than acting on. The RPE result follows uptake of exogenous synthetic peptide, so it shows import rather than synthesis in the organelle, and the antibody-based results cannot distinguish this accession from the nuclear MT-RNR2-like paralogs. Neither undermines the annotation, but neither establishes that humanin is made in the matrix. ORGANELLES check (2026-11-03). HSP60 double staining, immuno-EM and co-localisation of labelled peptide with mitochondria are organelle-level readouts, the peptide has no transit peptide or transmembrane segment, and no cached study fractionates humanin into a sub-compartment, so the rows stay at the root. Moved from ACCEPT to KEEP_AS_NON_CORE because humanin's characterised activities are extracellular (receptor ligand) or cytosolic (holding BAX and tBID away from mitochondria), which is what core_functions record; the mitochondrial pool is real but not where a core activity is shown. Supporting Evidence: PMID:15567815 Double staining with anti-humanin and anti-heat shock protein 60 showed that humanin was expressed mainly in mitochondria PMID:26990160 Exogenous HN was taken up by RPE and colocalized with mitochondria. |
| GO:0005739 mitochondrion | IDA PMID:26990160 The Mitochondrial-Derived Peptide Humanin Protects RPE Cells... | KEEP AS NON CORE | Summary: FITC-labelled humanin added to human RPE cells is taken up and colocalises with mitochondria. Reason: A mitochondrial pool is reported by three independent approaches and is unsurprising for a peptide whose ORF lies in the mitochondrial genome. Two caveats worth recording rather than acting on. The RPE result follows uptake of exogenous synthetic peptide, so it shows import rather than synthesis in the organelle, and the antibody-based results cannot distinguish this accession from the nuclear MT-RNR2-like paralogs. Neither undermines the annotation, but neither establishes that humanin is made in the matrix. ORGANELLES check (2026-11-03). HSP60 double staining, immuno-EM and co-localisation of labelled peptide with mitochondria are organelle-level readouts, the peptide has no transit peptide or transmembrane segment, and no cached study fractionates humanin into a sub-compartment, so the rows stay at the root. Moved from ACCEPT to KEEP_AS_NON_CORE because humanin's characterised activities are extracellular (receptor ligand) or cytosolic (holding BAX and tBID away from mitochondria), which is what core_functions record; the mitochondrial pool is real but not where a core activity is shown. Supporting Evidence: PMID:15567815 Double staining with anti-humanin and anti-heat shock protein 60 showed that humanin was expressed mainly in mitochondria PMID:26990160 Exogenous HN was taken up by RPE and colocalized with mitochondria. |
| GO:0005739 mitochondrion | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Electronic annotation from the UniProt subcellular location 'Mitochondrion' (UniProtKB-SubCell:SL-0173). Reason: Consistent with the experimental rows. ORGANELLES check (2026-11-03), examined as a synthesis row. SL-0173 rests on UniProt's curated location tied to the sperm immuno-EM and RPE uptake papers (ECO:0000269), so the organelle claim holds and the sources themselves never resolved a sub-compartment. Moved to KEEP_AS_NON_CORE to match the IDA rows. Supporting Evidence: file:human/MT-RNR2__Q8IVG9/MT-RNR2__Q8IVG9-uniprot.txt Also detected in sperm mitochondria (PubMed:20542501) PMID:15567815 Double staining with anti-humanin and anti-heat shock protein 60 showed that humanin was expressed mainly in mitochondria |
| GO:0006879 intracellular iron ion homeostasis | NAS PMID:15567815 Increased expression of humanin peptide in diffuse-type pigm... | REMOVE | Summary: Non-traceable author statement attached to the observation that humanin immunoreactivity in villonodular synovitis is seen predominantly around dense intramitochondrial iron deposits. Reason: Proximity of a peptide to iron deposits in a diseased tissue is not participation in iron homeostasis. No assay of iron uptake, storage, export or sensing was performed, no iron-related partner or activity has ever been reported for humanin, and the annotation is an NAS - the weakest evidence class - on a single descriptive pathology study. This is a clear over-annotation and should be withdrawn. Supporting Evidence: PMID:15567815 Electron microscopy disclosed immunolocalisation of this peptide, predominantly around dense iron deposits |
| GO:0007005 mitochondrion organization | IDA PMID:29432738 Humanin promotes mitochondrial biogenesis in pancreatic MIN6... | MARK AS OVER ANNOTATED | Summary: Exogenous humanin applied to MIN6 pancreatic beta-cells increases PPARGC1A/PGC1A expression and mitochondrial biogenesis markers. Reason: A transcriptional, several-steps-downstream consequence of receptor signalling by exogenously applied peptide in one cell line, read out as marker expression rather than as an effect of humanin on mitochondrial structure. Humanin performs none of the work of organising mitochondria: the step is executed by the PGC1A-driven biogenesis programme. Keeping the row would make a phenotype of peptide treatment look like an activity of the peptide. Supporting Evidence: PMID:29432738 Mitochondrial dysfunction is associated with |
| GO:0007267 cell-cell signaling | IDA PMID:12860203 Identification of essential amino acids in Humanin, a neurop... | ACCEPT | Summary: Humanin is secreted from expressing cells and acts on receptors of other cells; the secretion determinants were mapped in this study. Reason: A fair, if general, description of what humanin does: it is a secreted peptide that signals to receptors on other cells. The specific molecular function (receptor ligand activity) and pathway (STAT signalling) rows carry the detail. Supporting Evidence: PMID:12860203 Arg substitution revealed that the two structures-Leu9-Leu11 and Pro19-Va120-were essential for the secretion of full-length HN PMID:15465011 We have discovered that humanin (HN) acts as a ligand for formyl peptide receptor-like 1 (FPRL1) and 2 (FPRL2) |
| GO:0030595 leukocyte chemotaxis | IDA PMID:15153530 Humanin, a newly identified neuroprotective factor, uses the... | ACCEPT | Summary: Synthetic humanin induces chemotaxis of human monocytes and of FPRL1/FPR2-transfected cells, and the response is lost when the receptor is absent. Reason: Humanin itself performs the work of this process in the sense GO intends for a chemoattractant: it is the ligand whose receptor engagement directs the migration, which is how chemokines are annotated. The evidence is direct and receptor-resolved. Could be made more specific (monocyte chemotaxis), but the paper describes 'mononuclear phagocytes' and the general term is safe. Supporting Evidence: PMID:15153530 Humanin induced chemotaxis of mononuclear phagocytes by using a human G protein-coupled formylpeptide receptor-like-1 (FPRL1) and its murine counterpart FPR2 |
| GO:0031514 motile cilium | IEA GO_REF:0000044 | ACCEPT | Summary: Electronic annotation from the UniProt subcellular location 'Cilium, flagellum' (UniProtKB-SubCell:SL-0117), derived from the sperm flagellum immunolocalization. Reason: Consistent with the two independent sperm immunofluorescence studies; the sperm flagellum is a motile cilium, so this is the general form of the GO:0036126 and GO:0097225 rows. Supporting Evidence: PMID:20542501 HN is mainly localized in the midpiece of sperm in semen samples with normal morphology and in cytoplasmic residues and entire tail in those with abnormal morphology PMID:30920769 Humanin was expressed in the midpiece of the spermatozoa |
| GO:0032692 negative regulation of interleukin-1 production | IDA PMID:32923762 Humanin Ameliorates Free Fatty Acid-Induced Endothelial Infl... | MARK AS OVER ANNOTATED | Summary: In human endothelial cells treated with free fatty acids, exogenous humanin reduces TXNIP expression, NLRP3 inflammasome activation and consequently the expression of IL1B. Reason: The cytokine-production rows sit two steps downstream of anything humanin touches: humanin acts upstream of TXNIP and the inflammasome, and reduced IL-1beta/IL-18 is the readout of inflammasome suppression, not a separate activity. One HUVEC study with exogenous peptide generates five GOA rows; the mechanistically specific one (negative regulation of NLRP3 inflammasome complex assembly) carries the information and these two add a cell-type-specific phenotype instead. Supporting Evidence: PMID:32923762 Consequently, humanin inhibited the expression of IL-1 PMID:32923762 Moreover, the same doses of humanin inhibited the protein of TxNIP |
| GO:0032701 negative regulation of interleukin-18 production | IDA PMID:32923762 Humanin Ameliorates Free Fatty Acid-Induced Endothelial Infl... | MARK AS OVER ANNOTATED | Summary: In human endothelial cells treated with free fatty acids, exogenous humanin reduces TXNIP expression, NLRP3 inflammasome activation and consequently the expression of IL18. Reason: The cytokine-production rows sit two steps downstream of anything humanin touches: humanin acts upstream of TXNIP and the inflammasome, and reduced IL-1beta/IL-18 is the readout of inflammasome suppression, not a separate activity. One HUVEC study with exogenous peptide generates five GOA rows; the mechanistically specific one (negative regulation of NLRP3 inflammasome complex assembly) carries the information and these two add a cell-type-specific phenotype instead. Supporting Evidence: PMID:32923762 Consequently, humanin inhibited the expression of IL-1 PMID:32923762 Moreover, the same doses of humanin inhibited the protein of TxNIP |
| GO:0036126 sperm flagellum | IDA PMID:30920769 Humanin levels in human seminal plasma and spermatozoa are r... | ACCEPT | Summary: Immunofluorescence of human spermatozoa localises humanin to the midpiece, and seminal-plasma and sperm humanin levels correlate with sperm concentration and progressive motility. Reason: Replicated by two independent groups using different antibodies and methods, with ELISA quantification in 164 patients; the most specific and best-supported of the sperm localization rows. Supporting Evidence: PMID:30920769 Humanin was expressed in the midpiece of the spermatozoa PMID:20542501 HN is mainly localized in the midpiece of sperm in semen samples with normal morphology and in cytoplasmic residues and entire tail in those with abnormal morphology |
| GO:0042802 identical protein binding | IPI PMID:12787071 Two serine residues distinctly regulate the rescue function ... | ACCEPT | Summary: Humanin self-associates into a homodimer; Ser7 is required for dimerization and dimerization is required for neuroprotection, so the S7A peptide is inactive. Reason: Homodimerization is demonstrated and is functionally necessary rather than incidental, which is exactly the case where GO:0042802 is informative. Supporting Evidence: PMID:12787071 Multiple series of experiments indicated that Ser7 is necessary for self-dimerization of HN, which is essential for neuroprotection by this factor |
| GO:0043066 negative regulation of apoptotic process | IDA PMID:11327724 Mechanisms of neuroprotection by a novel rescue factor human... | ACCEPT | Summary: Humanin cDNA or synthetic peptide abolishes neuronal cytotoxicity caused by the Swedish mutant APP and by Abeta, acting on both the intracellular APP-triggered mechanism and Abeta toxicity. Reason: Suppression of apoptosis is humanin's core biological role and the single best-replicated claim about it, demonstrated across cell types, insults and laboratories, with a defined molecular mechanism (sequestration of BAX, BID and BimEL) and with loss-of-binding point mutants that lose protection. The general term is the right level for the aggregate of these results. Supporting Evidence: PMID:15661737 Synthetic HN peptide binds purified Bid and tBid in vitro and blocks tBid-induced release of cytochrome c and SMAC from isolated mitochondria PMID:15661735 HN also protects cells against apoptosis induced by BimEL but not BimL and BimS in gene transfection studies PMID:26990160 Humanin protected RPE cells from oxidative stress-induced cell death by STAT3 phosphorylation and inhibiting caspase-3 activation. |
| GO:0043066 negative regulation of apoptotic process | IDA PMID:15661735 Cytoprotective peptide humanin binds and inhibits proapoptot... | ACCEPT | Summary: Humanin protects cells from BimEL-induced apoptosis and blocks BimEL-induced SMAC and cytochrome c release from bax-/- mitochondria; non-binding humanin mutants do not protect. Reason: Suppression of apoptosis is humanin's core biological role and the single best-replicated claim about it, demonstrated across cell types, insults and laboratories, with a defined molecular mechanism (sequestration of BAX, BID and BimEL) and with loss-of-binding point mutants that lose protection. The general term is the right level for the aggregate of these results. Supporting Evidence: PMID:15661737 Synthetic HN peptide binds purified Bid and tBid in vitro and blocks tBid-induced release of cytochrome c and SMAC from isolated mitochondria PMID:15661735 HN also protects cells against apoptosis induced by BimEL but not BimL and BimS in gene transfection studies PMID:26990160 Humanin protected RPE cells from oxidative stress-induced cell death by STAT3 phosphorylation and inhibiting caspase-3 activation. |
| GO:0043066 negative regulation of apoptotic process | IDA PMID:15661737 Humanin binds and nullifies Bid activity by blocking its act... | ACCEPT | Summary: Humanin blocks tBID-induced release of cytochrome c and SMAC from isolated mitochondria and protects intact cells from tBID overexpression; the inactive humanin mutant does not. Reason: Suppression of apoptosis is humanin's core biological role and the single best-replicated claim about it, demonstrated across cell types, insults and laboratories, with a defined molecular mechanism (sequestration of BAX, BID and BimEL) and with loss-of-binding point mutants that lose protection. The general term is the right level for the aggregate of these results. Supporting Evidence: PMID:15661737 Synthetic HN peptide binds purified Bid and tBid in vitro and blocks tBid-induced release of cytochrome c and SMAC from isolated mitochondria PMID:15661735 HN also protects cells against apoptosis induced by BimEL but not BimL and BimS in gene transfection studies PMID:26990160 Humanin protected RPE cells from oxidative stress-induced cell death by STAT3 phosphorylation and inhibiting caspase-3 activation. |
| GO:0043066 negative regulation of apoptotic process | IDA PMID:26990160 The Mitochondrial-Derived Peptide Humanin Protects RPE Cells... | ACCEPT | Summary: Humanin protects retinal pigment epithelium cells from oxidative-stress-induced death, with STAT3 phosphorylation and reduced caspase-3 activation. Reason: Suppression of apoptosis is humanin's core biological role and the single best-replicated claim about it, demonstrated across cell types, insults and laboratories, with a defined molecular mechanism (sequestration of BAX, BID and BimEL) and with loss-of-binding point mutants that lose protection. The general term is the right level for the aggregate of these results. Supporting Evidence: PMID:15661737 Synthetic HN peptide binds purified Bid and tBid in vitro and blocks tBid-induced release of cytochrome c and SMAC from isolated mitochondria PMID:15661735 HN also protects cells against apoptosis induced by BimEL but not BimL and BimS in gene transfection studies PMID:26990160 Humanin protected RPE cells from oxidative stress-induced cell death by STAT3 phosphorylation and inhibiting caspase-3 activation. |
| GO:0043066 negative regulation of apoptotic process | IDA PMID:27783653 Humanin Protects RPE Cells from Endoplasmic Reticulum Stress... | ACCEPT | Summary: Humanin protects RPE cells from ER-stress-induced apoptosis, restoring mitochondrial glutathione and lowering mitochondrial superoxide. Reason: Suppression of apoptosis is humanin's core biological role and the single best-replicated claim about it, demonstrated across cell types, insults and laboratories, with a defined molecular mechanism (sequestration of BAX, BID and BimEL) and with loss-of-binding point mutants that lose protection. The general term is the right level for the aggregate of these results. Supporting Evidence: PMID:15661737 Synthetic HN peptide binds purified Bid and tBid in vitro and blocks tBid-induced release of cytochrome c and SMAC from isolated mitochondria PMID:15661735 HN also protects cells against apoptosis induced by BimEL but not BimL and BimS in gene transfection studies PMID:26990160 Humanin protected RPE cells from oxidative stress-induced cell death by STAT3 phosphorylation and inhibiting caspase-3 activation. |
| GO:0043524 negative regulation of neuron apoptotic process | IDA PMID:12787071 Two serine residues distinctly regulate the rescue function ... | ACCEPT | Summary: Alanine and D-serine substitution series in neuronal rescue assays define the residues required for neuroprotection and show that Ser7-dependent dimerization is essential. Reason: The neuron-specific form is the founding and most thoroughly dissected activity of humanin: it was discovered in a screen for protection of neurons against familial AD genes, the sequence requirements were mapped residue by residue, and the receptor and STAT3 dependence were established in neuronal cells. A core biological process for this gene. Supporting Evidence: PMID:11371646 A rescue factor abolishing neuronal cell death by a wide spectrum of familial Alzheimer's disease genes and Abeta. PMID:16005025 We simultaneously provide evidence that neuroprotection by HN in F11 cells is mediated by the STAT3 transcription factor as well as by certain tyrosine kinases PMID:19386761 Overexpression of ciliary neurotrophic factor receptor alpha (CNTFR) and/or the IL-27 receptor subunit, WSX-1, but not that of any other tested gp130-related receptor subunit, up-regulated HN binding to neuronal cells, whereas siRNA-mediated knockdown of endogenous CNTFR and/or WSX-1 reduced it |
| GO:0043524 negative regulation of neuron apoptotic process | IDA PMID:16005025 Involvement of tyrosine kinases and STAT3 in Humanin-mediate... | ACCEPT | Summary: Neuroprotection by humanin in F11 neurohybrid cells requires STAT3 and tyrosine kinase activity, and is not abolished by knockdown of FPR2. Reason: The neuron-specific form is the founding and most thoroughly dissected activity of humanin: it was discovered in a screen for protection of neurons against familial AD genes, the sequence requirements were mapped residue by residue, and the receptor and STAT3 dependence were established in neuronal cells. A core biological process for this gene. Supporting Evidence: PMID:11371646 A rescue factor abolishing neuronal cell death by a wide spectrum of familial Alzheimer's disease genes and Abeta. PMID:16005025 We simultaneously provide evidence that neuroprotection by HN in F11 cells is mediated by the STAT3 transcription factor as well as by certain tyrosine kinases PMID:19386761 Overexpression of ciliary neurotrophic factor receptor alpha (CNTFR) and/or the IL-27 receptor subunit, WSX-1, but not that of any other tested gp130-related receptor subunit, up-regulated HN binding to neuronal cells, whereas siRNA-mediated knockdown of endogenous CNTFR and/or WSX-1 reduced it |
| GO:0043524 negative regulation of neuron apoptotic process | IDA PMID:19386761 Humanin inhibits neuronal cell death by interacting with a c... | ACCEPT | Summary: Humanin protects neuronal cells from AD-related insults through a gp130-family receptor complex involving CNTFR and WSX-1. Reason: The neuron-specific form is the founding and most thoroughly dissected activity of humanin: it was discovered in a screen for protection of neurons against familial AD genes, the sequence requirements were mapped residue by residue, and the receptor and STAT3 dependence were established in neuronal cells. A core biological process for this gene. Supporting Evidence: PMID:11371646 A rescue factor abolishing neuronal cell death by a wide spectrum of familial Alzheimer's disease genes and Abeta. PMID:16005025 We simultaneously provide evidence that neuroprotection by HN in F11 cells is mediated by the STAT3 transcription factor as well as by certain tyrosine kinases PMID:19386761 Overexpression of ciliary neurotrophic factor receptor alpha (CNTFR) and/or the IL-27 receptor subunit, WSX-1, but not that of any other tested gp130-related receptor subunit, up-regulated HN binding to neuronal cells, whereas siRNA-mediated knockdown of endogenous CNTFR and/or WSX-1 reduced it |
| GO:0048018 receptor ligand activity | IDA PMID:19386761 Humanin inhibits neuronal cell death by interacting with a c... | ACCEPT | Summary: Humanin binds neuronal cells through a cytokine receptor complex containing CNTFR, WSX-1/IL27RA and gp130, and receptor subunit overexpression and knockdown change both binding and neuroprotection. Reason: This is humanin's central molecular function and the one that explains most of its biology: a secreted peptide that engages cell-surface receptors - the gp130-family complex here and FPR2/FPR3 elsewhere - and changes their signalling output. Direct binding and receptor-dependence data support it. Supporting Evidence: PMID:19386761 Overexpression of ciliary neurotrophic factor receptor alpha (CNTFR) and/or the IL-27 receptor subunit, WSX-1, but not that of any other tested gp130-related receptor subunit, up-regulated HN binding to neuronal cells, whereas siRNA-mediated knockdown of endogenous CNTFR and/or WSX-1 reduced it PMID:15465011 We have discovered that humanin (HN) acts as a ligand for formyl peptide receptor-like 1 (FPRL1) and 2 (FPRL2) |
| GO:0048019 receptor antagonist activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic propagation of receptor antagonist activity from the PANTHER humanin-family node PTN002141596; humanin's own experimental annotation is among the descendant evidences behind the node, which is expected rather than circular. Reason: The IBA follows mechanically from descent from a node whose placement rests on the humanin FPR2/Abeta competition data. There is no target-specific evidence of loss or divergence to argue with - humanin is the founding member of the clade - so the node placement stands, and the IBA says the function is inherited across the humanin family rather than specific to this ORF. The same caveat as the IDA row applies: the antagonism is of amyloid-beta at FPR2, not of the receptor's own signalling. Supporting Evidence: PMID:15153530 Humanin induced chemotaxis of mononuclear phagocytes by using a human G protein-coupled formylpeptide receptor-like-1 (FPRL1) and its murine counterpart FPR2 |
| GO:0048019 receptor antagonist activity | IDA PMID:15153530 Humanin, a newly identified neuroprotective factor, uses the... | ACCEPT | Summary: Humanin competitively inhibits access of amyloid-beta 42 to FPR2/FPRL1 on mononuclear phagocytes, reducing Abeta42-induced responses, while itself acting as an agonist of the same receptor. Reason: The term definition - interacting with a receptor to decrease the ability of the receptor agonist to bind and activate it - is met: humanin occupies FPR2 and displaces Abeta42 from it. Worth noting that humanin is simultaneously an FPR2 agonist (it triggers chemotaxis and suppresses cAMP through the same receptor), so the antagonist and ligand annotations are describing competition rather than silent occupancy. Not a case for removal: the curator's reading matches the definition and the data. Supporting Evidence: PMID:15153530 Humanin induced chemotaxis of mononuclear phagocytes by using a human G protein-coupled formylpeptide receptor-like-1 (FPRL1) and its murine counterpart FPR2 PMID:15465011 We have discovered that humanin (HN) acts as a ligand for formyl peptide receptor-like 1 (FPRL1) and 2 (FPRL2) |
| GO:0048471 perinuclear region of cytoplasm | IDA PMID:15619032 Interaction of the spectrin-like repeats of alpha-actinin-4 ... | MARK AS OVER ANNOTATED | Summary: GFP-fused humanin overexpressed in HEK293 cells colocalises with endogenous alpha-actinin mainly in the perinuclear cytoplasm. Reason: A single observation from an overexpressed GFP fusion in a heterologous cell line, reported incidentally in a study about actinin-4 ligands, with no functional correlate. The generic cytoplasm annotations already cover the intracellular pool; this adds a spurious degree of spatial precision. The companion ACTN4 protein-binding row is likewise recommended for removal. Supporting Evidence: PMID:15619032 Green fluorescent protein-fused humanin and endogenous actinin colocalized mainly in the perinuclear cytoplasm of HEK293 cells |
| GO:0050728 negative regulation of inflammatory response | IDA PMID:32923762 Humanin Ameliorates Free Fatty Acid-Induced Endothelial Infl... | KEEP AS NON CORE | Summary: Exogenous humanin reduces free-fatty-acid-induced inflammation in endothelial cells, acting through suppression of TXNIP and the NLRP3 inflammasome. Reason: A genuine, mechanistically traced anti-inflammatory effect, but one of several tissue-level cytoprotective phenotypes that follow from receptor signalling by exogenously applied peptide; it is not the gene's core function and should not be read as a distinct activity. Retained at this general level in preference to the two cytokine-production rows from the same study. Supporting Evidence: PMID:32923762 Humanin Ameliorates Free Fatty Acid-Induced Endothelial Inflammation by Suppressing the NLRP3 Inflammasome. PMID:32923762 Moreover, the same doses of humanin inhibited the protein of TxNIP |
| GO:0097225 sperm midpiece | IDA PMID:20542501 Immunolocalization of humanin in human sperm and testis. | ACCEPT | Summary: Immunoelectron microscopy localises humanin to the sperm midpiece, where it is highly concentrated. Reason: Replicated by two groups with different methods; the most specific sperm localization available and consistent with the mitochondria-rich midpiece. Its functional meaning is unresolved, which is recorded as a suggested question rather than acted on. Supporting Evidence: PMID:20542501 HN is mainly localized in the midpiece of sperm in semen samples with normal morphology and in cytoplasmic residues and entire tail in those with abnormal morphology PMID:30920769 Humanin was expressed in the midpiece of the spermatozoa |
| GO:0097225 sperm midpiece | IDA PMID:30920769 Humanin levels in human seminal plasma and spermatozoa are r... | ACCEPT | Summary: Immunofluorescence confirms humanin in the midpiece of human spermatozoa in a 164-patient cohort. Reason: Replicated by two groups with different methods; the most specific sperm localization available and consistent with the mitochondria-rich midpiece. Its functional meaning is unresolved, which is recorded as a suggested question rather than acted on. Supporting Evidence: PMID:20542501 HN is mainly localized in the midpiece of sperm in semen samples with normal morphology and in cytoplasmic residues and entire tail in those with abnormal morphology PMID:30920769 Humanin was expressed in the midpiece of the spermatozoa |
| GO:0097435 supramolecular fiber organization | IDA PMID:31690630 Humanin induces conformational changes in the apoptosis regu... | ACCEPT | Summary: Recombinant humanin and BAX form fibers together in vitro; BAX undergoes secondary and tertiary structural rearrangement and is incorporated into the fibers, and humanin mutations that alter anti-apoptotic activity change fiber morphology. Reason: Humanin does part of the work here rather than merely being required for it: it co-polymerises into the fiber, and point mutants that change its activity change the fiber. This is the proposed mechanism behind the BAX and BID binding and anti-apoptotic annotations. The caveat to record is that the work is entirely in vitro with purified recombinant proteins at high concentration, so whether these fibers form in cells is unknown. Supporting Evidence: PMID:31690630 we report that HN and BAX can form fibers together in vitro PMID:33106313 BID fibers are similar to those produced using BAX; however, the structures differ in final conformations of the BCL-2 proteins. |
| GO:0097435 supramolecular fiber organization | IDA PMID:33106313 Humanin selectively prevents the activation of pro-apoptotic... | ACCEPT | Summary: Humanin and BID likewise co-assemble into fibers, characterised by spectroscopy, protease fragmentation and electron microscopy; the BID fibers differ from the BAX fibers in the final conformation of the BCL-2 protein. Reason: Humanin does part of the work here rather than merely being required for it: it co-polymerises into the fiber, and point mutants that change its activity change the fiber. This is the proposed mechanism behind the BAX and BID binding and anti-apoptotic annotations. The caveat to record is that the work is entirely in vitro with purified recombinant proteins at high concentration, so whether these fibers form in cells is unknown. Supporting Evidence: PMID:31690630 we report that HN and BAX can form fibers together in vitro PMID:33106313 BID fibers are similar to those produced using BAX; however, the structures differ in final conformations of the BCL-2 proteins. |
| GO:0097696 cell surface receptor signaling pathway via STAT | IDA PMID:16005025 Involvement of tyrosine kinases and STAT3 in Humanin-mediate... | ACCEPT | Summary: Humanin-mediated neuroprotection requires STAT3 and tyrosine kinase activity, placing humanin upstream of a STAT-dependent cell-surface receptor signalling pathway; the annotation uses acts_upstream_of_positive_effect. Reason: Correct and correctly qualified: humanin is the extracellular ligand, so it acts upstream of the pathway rather than executing it, and the qualifier says so. Independently supported by the gp130/CNTFR receptor complex work and by STAT3 phosphorylation in RPE cells. Supporting Evidence: PMID:16005025 We simultaneously provide evidence that neuroprotection by HN in F11 cells is mediated by the STAT3 transcription factor as well as by certain tyrosine kinases PMID:26990160 Humanin protected RPE cells from oxidative stress-induced cell death by STAT3 phosphorylation and inhibiting caspase-3 activation. PMID:19386761 Overexpression of ciliary neurotrophic factor receptor alpha (CNTFR) and/or the IL-27 receptor subunit, WSX-1, but not that of any other tested gp130-related receptor subunit, up-regulated HN binding to neuronal cells, whereas siRNA-mediated knockdown of endogenous CNTFR and/or WSX-1 reduced it |
| GO:0150079 negative regulation of neuroinflammatory response | IDA PMID:23277413 Neuroprotective Peptide humanin inhibits inflammatory respon... | KEEP AS NON CORE | Summary: Humanin at 5-20 micromolar inhibits LPS-induced astrocyte activation and lowers IL-6, IL-1beta and TNF-alpha in cultured astrocytes, though it does not fully reverse the response. Reason: A real but partial effect, shown in one cell-culture model with exogenous peptide at micromolar concentrations - well above the nanomolar potencies measured at FPR2 - and without a defined mechanism. Plausibly a consequence of the same receptor signalling that underlies the anti-apoptotic function; not a core function of the gene. Supporting Evidence: PMID:23277413 We also found pretreatment with HN decreased the level of proinflammatory cytokines, interleukin (IL)-6, IL-1 PMID:23277413 we noticed HN couldn't completely reverse the above inflammatory injury |
| GO:1900118 negative regulation of execution phase of apoptosis | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic propagation from the humanin-family node PTN002141596, with a humanin-like paralog (UniProtKB:P0CJ72) among the descendant evidences. Reason: The node placement is sound and the term fits the mechanism: by blocking BAX/BAK oligomerization and the release of cytochrome c and SMAC, humanin prevents the caspase activation that constitutes the execution phase. More specific than, and consistent with, the GO:0043066 rows. The humanin family is the clade in which this activity was characterised, so there is no node-placement objection to raise. Supporting Evidence: PMID:15661737 Synthetic HN peptide binds purified Bid and tBid in vitro and blocks tBid-induced release of cytochrome c and SMAC from isolated mitochondria PMID:15661735 HN also protects cells against apoptosis induced by BimEL but not BimL and BimS in gene transfection studies |
| GO:1900226 negative regulation of NLRP3 inflammasome complex assembly | IDA PMID:32923762 Humanin Ameliorates Free Fatty Acid-Induced Endothelial Infl... | KEEP AS NON CORE | Summary: Humanin reduces free-fatty-acid-induced TXNIP expression and NLRP3 inflammasome activation in endothelial cells, with reduced NLRP3, ASC and caspase-1 downstream output. Reason: The most mechanistically specific of the five rows generated by this single HUVEC study, and the one worth keeping. Retained as non-core: it is an effect of exogenously applied peptide in one cell type, and humanin acts upstream of the inflammasome rather than being part of it. Supporting Evidence: PMID:32923762 Moreover, the same doses of humanin inhibited the protein of TxNIP PMID:32923762 Humanin Ameliorates Free Fatty Acid-Induced Endothelial Inflammation by Suppressing the NLRP3 Inflammasome. |
| GO:1902883 negative regulation of response to oxidative stress | IDA PMID:32923762 Humanin Ameliorates Free Fatty Acid-Induced Endothelial Infl... | MARK AS OVER ANNOTATED | Summary: In the same endothelial study, humanin lowers markers of oxidative stress accompanying free-fatty-acid treatment. Reason: Reduced oxidative-stress markers in cells rescued from an inflammatory insult is a phenotype of protection, not a regulated process humanin participates in, and no humanin activity on any antioxidant or ROS-generating step has been identified. The correctly-aimed rows from this study are the inflammasome and inflammatory-response terms. Supporting Evidence: PMID:32923762 Moreover, the same doses of humanin inhibited the protein of TxNIP PMID:27783653 TM treatment increased mitochondrial superoxide production, and HN co-treatment resulted in a decrease in mitochondrial superoxide compared to TM treatment alone. |
| GO:1904646 cellular response to amyloid-beta | IDA PMID:12787071 Two serine residues distinctly regulate the rescue function ... | ACCEPT | Summary: Humanin rescues neuronal cells from amyloid-beta toxicity, and the sequence requirements for this rescue were mapped. Reason: Core: responding to amyloid-beta by protecting the cell is the activity humanin was discovered through, it is reproduced across laboratories, and both the receptor arm and the direct Abeta-binding arm of the mechanism are documented. Supporting Evidence: PMID:11371646 A rescue factor abolishing neuronal cell death by a wide spectrum of familial Alzheimer's disease genes and Abeta. PMID:11327724 Transfection of neuronal cells with HN cDNA or treatment with the coding HN polypeptide abrogated cytotoxicity by NL-APP PMID:19386761 Overexpression of ciliary neurotrophic factor receptor alpha (CNTFR) and/or the IL-27 receptor subunit, WSX-1, but not that of any other tested gp130-related receptor subunit, up-regulated HN binding to neuronal cells, whereas siRNA-mediated knockdown of endogenous CNTFR and/or WSX-1 reduced it |
| GO:1904646 cellular response to amyloid-beta | IDA PMID:19386761 Humanin inhibits neuronal cell death by interacting with a c... | ACCEPT | Summary: Humanin protection against amyloid-beta and AD-gene insults in neuronal cells is mediated by the CNTFR/WSX-1/gp130 receptor complex. Reason: Core: responding to amyloid-beta by protecting the cell is the activity humanin was discovered through, it is reproduced across laboratories, and both the receptor arm and the direct Abeta-binding arm of the mechanism are documented. Supporting Evidence: PMID:11371646 A rescue factor abolishing neuronal cell death by a wide spectrum of familial Alzheimer's disease genes and Abeta. PMID:11327724 Transfection of neuronal cells with HN cDNA or treatment with the coding HN polypeptide abrogated cytotoxicity by NL-APP PMID:19386761 Overexpression of ciliary neurotrophic factor receptor alpha (CNTFR) and/or the IL-27 receptor subunit, WSX-1, but not that of any other tested gp130-related receptor subunit, up-regulated HN binding to neuronal cells, whereas siRNA-mediated knockdown of endogenous CNTFR and/or WSX-1 reduced it |
| GO:1905907 negative regulation of amyloid fibril formation | IDA PMID:27349871 Solution NMR structure and inhibitory effect against amyloid... | ACCEPT | Summary: Humanin with D-Ser14 binds amyloid-beta with high affinity and potently inhibits its fibrillation; the solution NMR structure of the peptide was determined in the same study. Reason: Humanin does the work of this process directly: it binds amyloid-beta and, by doing so, slows fibril assembly in cell-free systems. This is a genuine molecular activity rather than a downstream phenotype, and it is paired with the GO:0001540 amyloid-beta binding replacement proposed for the APP interaction row. Supporting Evidence: PMID:27349871 we demonstrated that Humanin d-Ser14 exhibited potent inhibitory activity against fibrillation of amyloid- PMID:28282805 Thioflavin-T assay indicated that both HN and HNG delay the formation and reduce the final amount of PMID:28282805 In vitro surface plasmon resonance studies indicated that they interact with |
| GO:1905907 negative regulation of amyloid fibril formation | IDA PMID:28282805 Humanin Specifically Interacts with Amyloid-Ξ² Oligomers and ... | ACCEPT | Summary: Surface plasmon resonance shows humanin and HNG interact with soluble Abeta42 oligomers, and thioflavin-T assays show both delay fibril formation and reduce the final amount of fibrils; the peptides also reduce Abeta oligomer toxicity in a transgenic C. elegans model. Reason: Humanin does the work of this process directly: it binds amyloid-beta and, by doing so, slows fibril assembly in cell-free systems. This is a genuine molecular activity rather than a downstream phenotype, and it is paired with the GO:0001540 amyloid-beta binding replacement proposed for the APP interaction row. Supporting Evidence: PMID:27349871 we demonstrated that Humanin d-Ser14 exhibited potent inhibitory activity against fibrillation of amyloid- PMID:28282805 Thioflavin-T assay indicated that both HN and HNG delay the formation and reduce the final amount of PMID:28282805 In vitro surface plasmon resonance studies indicated that they interact with |
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Download this section (compressed HTML)Q: Is the biologically active humanin peptide the product of the mitochondrial MT-RNR2 ORF or of one of the thirteen nuclear MT-RNR2-like (NUMT-derived) loci? Every antibody-based detection of humanin in tissue, and therefore several of the localization annotations on this entry, cannot distinguish the two. GO curation of this accession would benefit from an explicit convention for peptides whose coding assignment is unresolved.
Q: If humanin is translated from the mitochondrial transcript inside the organelle, the mitochondrial genetic code yields a 21-residue peptide lacking Lys-Arg-Arg-Ala. Which length is the physiological species, and do the published structure-activity data (all obtained with the 24-mer or the S14G 24-mer) apply to it?
Q: How does a peptide encoded in the mitochondrial genome reach the extracellular space? Secretion is well documented and its sequence determinants are mapped, but no export route from the matrix, and no cytoplasmic translation mechanism for an mtDNA-encoded mRNA, has been demonstrated.
Q: Do the humanin-BAX and humanin-BID fibers characterised in vitro with purified recombinant proteins exist in cells, and at what stoichiometry? This is the mechanistic basis of the anti-apoptotic annotations and has so far only been shown cell-free.
Q: Which receptor system accounts for which phenotype? FPR2 knockdown does not abolish humanin neuroprotection in F11 cells (PMID:16005025), whereas the CNTFR/WSX-1/gp130 complex is required (PMID:19386761); several of the cytoprotective process annotations are not attributed to either receptor.
Q: Does humanin have a function in sperm? Localization to the flagellar midpiece is replicated and seminal-plasma levels track sperm concentration and motility, but no causal experiment exists, and the sperm annotations are purely locational.
Q: GO has no term for binding an insulin-like growth factor binding protein, so the three humanin-IGFBP3 rows sit as bare protein binding. Should such a term be created (see proposed_new_terms), or should the interaction be captured on IGFBP3 instead?
Experiment: Targeted mass spectrometry of purified human plasma, mitochondria and cytosol, using heavy-labelled 24-mer and 21-mer standards and peptides diagnostic for the mitochondrial sequence versus each MTRNR2L paralog, to establish which peptide species exists and where.
Hypothesis: The circulating cytoprotective peptide is distinguishable by mass and sequence from the mitochondrially-coded 21-mer.
Experiment: Ribosome profiling and mitoribosome profiling of the MT-RNR2 locus, combined with a cytoplasmic versus mitochondrial translation-inhibitor split (cycloheximide versus chloramphenicol), to determine which ribosome population translates the humanin ORF.
Hypothesis: The humanin ORF is translated by cytoplasmic ribosomes from an exported or nuclear-encoded transcript.
Experiment: Base-edit or delete the humanin ORF in the mitochondrial genome (mitochondrial base editing) and separately knock out the expressed MTRNR2L loci, then assay apoptotic sensitivity, STAT3 activation and circulating peptide levels, to attribute the phenotype to a locus.
Hypothesis: Loss-of-function at one locus class, not the other, abolishes the cytoprotective activity.
Experiment: Crosslinking or FRET detection of humanin-BAX and humanin-BID assemblies in intact cells at endogenous peptide concentrations, with the fiber-altering humanin point mutants (C8A, C8P, S14G) as controls.
Hypothesis: Humanin-BCL-2-family fibers form in cells and their abundance tracks anti-apoptotic potency.
Experiment: Dose-response comparison of wild-type humanin against the S14G analogue across the published cytoprotective assays at nanomolar concentrations matching the measured FPR2 EC50, to establish which reported phenotypes occur at physiological peptide levels.
Hypothesis: Several of the downstream cytoprotective phenotypes require micromolar peptide and do not occur at circulating concentrations.
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