{"gene":"AMPH","run_date":"2026-06-09T22:02:43","timeline":{"discoveries":[{"year":2009,"finding":"C. elegans AMPH-1 (Amphiphysin/BIN1 family, BAR-domain protein) colocalizes with RME-1 on recycling endosomes in vivo; AMPH-1 NPF/D/E sequences bind the RME-1 EH-domain; deletion of amph-1 causes defects in recycling endosome morphology and cargo recycling; purified recombinant AMPH-1–RME-1 complexes produce short coated membrane tubules distinct from those produced by either protein alone, indicating cooperative regulation of endocytic recycling.","method":"In vivo colocalization (live imaging), deletion mutant phenotypic analysis, in vitro reconstitution of membrane tubulation with purified proteins, EH-domain binding assay","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro reconstitution with purified proteins, genetic loss-of-function with defined cellular phenotype, direct binding assay, and functional rescue; multiple orthogonal methods in one study","pmids":["19915558"],"is_preprint":false},{"year":2015,"finding":"RAB-10/Rab10 and AMPH-1/Amphiphysin directly bind the RAB-5 GAP TBC-2 and recruit it to endosomes; in the absence of RAB-10 or AMPH-1 binding to TBC-2, RAB-5 membrane association is abnormally elevated and recycling cargo is trapped in early endosomes, establishing that AMPH-1–mediated downregulation of RAB-5 is required for cargo exit from early endosomes.","method":"Binding assays (Co-IP/pulldown), genetic loss-of-function (deletion mutants), fluorescence colocalization in vivo, epistasis analysis","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal binding demonstrated, genetic epistasis with defined cargo-recycling phenotype, multiple orthogonal methods in one study","pmids":["26393361"],"is_preprint":false},{"year":2022,"finding":"Purified C. elegans AMPH-1 alone is sufficient to drive membrane fission, converting large unilamellar vesicles into small tubular-vesicular products; this fission requires the amphipathic H0 helix of AMPH-1; RME-1 slows fission; unexpectedly, GTP stimulates AMPH-1-induced membrane fission. The yeast heterodimeric N-BAR protein Rvs161/167p shows the same GTP-stimulated fission, suggesting this is a general property of N-BAR proteins.","method":"In vitro membrane fission assay using burst analysis spectroscopy (BAS) with purified protein, H0-helix mutant analysis, GTP addition experiment, comparative assay with yeast Rvs161/167p","journal":"Traffic (Copenhagen, Denmark)","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstitution with purified protein, mutagenesis of H0 helix, multiple conditions tested in one study; single lab but orthogonal methods","pmids":["36435193"],"is_preprint":false},{"year":2025,"finding":"GTP binding stabilizes interactions between AMPH-1 and the membrane through amphipathic N-terminal α-helices at the tips of the arc-shaped homodimeric structure; GDP-bound (post-hydrolysis) AMPH-1 repositions these helices to interact with helices of other homodimers, forming an oligomeric AMPH-1 lattice that tubulates membranes in preparation for carrier formation by membrane fission.","method":"Structural and biochemical characterization of GTP- vs. GDP-bound states, liposome tubulation assays, mutational analysis of N-terminal helices","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 1 / Moderate — structural model linked to functional membrane-tubulation assay with mutagenesis, single lab but multiple orthogonal methods","pmids":["40749062"],"is_preprint":false},{"year":2026,"finding":"In Drosophila muscle, LUBEL/RNF31 (a ubiquitin E3 ligase for linear/M1-linked ubiquitination) directly interacts with Amphiphysin (Amph, a BAR-domain protein); LUBEL ubiquitin ligase activity and the LUBEL–Amph interaction are required for proper T-tubule morphology; loss of LUBEL produces Amph-positive membrane sheets instead of tubular networks; LUBEL and M1-linked ubiquitin chains assemble into puncta on membranes through multivalent interactions, facilitating Amph-mediated membrane tubulation. The Amph–LUBEL/RNF31 interaction is evolutionarily conserved.","method":"Genetic loss-of-function (LUBEL mutants in Drosophila), Co-IP/direct interaction assay, fluorescence microscopy of T-tubule morphology, ubiquitin ligase activity assays","journal":"Science advances","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal interaction demonstrated, genetic loss-of-function with defined morphological phenotype, multiple methods; Drosophila ortholog study, conserved interaction noted but single lab","pmids":["41499502"],"is_preprint":false},{"year":1995,"finding":"Human amphiphysin (AMPH) is peripherally associated with synaptic vesicles; it is expressed in neurons, certain endocrine cell types, and spermatocytes; the gene was mapped to chromosome 7p13-p14; autoantibodies against amphiphysin occur in paraneoplastic Stiff-Man syndrome.","method":"cDNA cloning, primary structure determination, chromosomal mapping, immunolocalization","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct subcellular localization by biochemical fractionation (synaptic vesicle association), cDNA-level primary structure, replicated in human and chicken; no functional mutagenesis","pmids":["7757077"],"is_preprint":false},{"year":2018,"finding":"Knockdown of AMPH-1 in lung cancer cells activates the Ras-Raf-MEK-ERK signaling pathway, promoting cell proliferation, attenuating apoptosis, and accelerating cell cycle progression; overexpression reverses these effects, placing AMPH-1 as a negative regulator of this pathway.","method":"shRNA knockdown and overexpression in lung cancer cell lines, western blot for ERK pathway components, in vivo xenograft mouse model","journal":"Lasers in medical science","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single KD/OE approach with pathway readout but no direct binding or reconstitution demonstrating mechanism; pathway placement is correlative","pmids":["30143925"],"is_preprint":false},{"year":2018,"finding":"Knockdown of AMPH-1 in breast cancer cells activates ERK and EMT pathways, promoting proliferation, migration, and cell cycle progression while attenuating apoptosis, consistent with AMPH-1 acting as a negative regulator of ERK and EMT signaling.","method":"shRNA knockdown in breast cancer cell lines, western blot for ERK/EMT markers, proliferation/migration assays, in vivo xenograft","journal":"Journal of Cancer","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, pathway placement by KD with no direct molecular interaction demonstrated","pmids":["29937937"],"is_preprint":false}],"current_model":"AMPH/Amphiphysin is a BAR-domain (N-BAR) protein that uses its amphipathic H0 helix to sense and deform membranes; GTP binding stabilizes its membrane interaction through these N-terminal helices, while GTP hydrolysis drives repositioning of the helices to form an oligomeric lattice that tubulates membranes, ultimately enabling membrane fission to produce transport carriers from the recycling endosome; it cooperates with RME-1/EHD1 (binding via NPF sequences to the EH-domain) to recycle transmembrane cargo, and promotes endosomal maturation by recruiting the RAB-5 GAP TBC-2 together with RAB-10 to downregulate RAB-5 and allow cargo exit from early endosomes; in muscle, its membrane-tubulating activity is regulated by linear ubiquitination via the LUBEL/RNF31 E3 ligase, which is required for T-tubule biogenesis."},"narrative":{"mechanistic_narrative":"AMPH/Amphiphysin is an N-BAR-domain protein that senses and remodels membranes to generate transport carriers during endocytic recycling and membrane-tubule biogenesis [PMID:19915558, PMID:36435193]. Purified AMPH-1 is sufficient to deform membranes and drive fission, converting vesicles into tubular-vesicular products in a reaction that depends on its amphipathic H0 helix and is stimulated by GTP [PMID:36435193]. GTP binding stabilizes membrane contacts through the N-terminal helices at the tips of the arc-shaped homodimer, and post-hydrolysis (GDP-bound) repositioning of these helices drives assembly of an oligomeric AMPH-1 lattice that tubulates the membrane prior to fission [PMID:40749062]. In endocytic recycling, AMPH-1 colocalizes with and binds the EH-domain of RME-1/EHD1 through its NPF/D/E motifs, and the two proteins cooperatively produce coated membrane tubules and are required for recycling-endosome morphology and cargo recycling [PMID:19915558]; AMPH-1 further promotes cargo exit from early endosomes by binding RAB-10 and recruiting the RAB-5 GAP TBC-2 to downregulate RAB-5 [PMID:26393361]. In muscle, AMPH-mediated tubulation underlies T-tubule biogenesis and is controlled by linear (M1-linked) ubiquitination through direct interaction with the E3 ligase LUBEL/RNF31, whose activity is required to convert Amph-positive membrane sheets into tubular networks [PMID:41499502]. AMPH was originally identified as a synaptic-vesicle-associated neuronal protein and the autoantigen in paraneoplastic Stiff-Man syndrome [PMID:7757077].","teleology":[{"year":2009,"claim":"Established that AMPH-1 acts in endocytic recycling by partnering with RME-1/EHD1, answering whether this BAR protein functions beyond synaptic endocytosis.","evidence":"In vivo colocalization, amph-1 deletion phenotypes, EH-domain binding assay, and in vitro tubulation with purified AMPH-1–RME-1 in C. elegans","pmids":["19915558"],"confidence":"High","gaps":["Did not resolve the structural basis of tubulation","Cargo specificity and the full repertoire of recycled transmembrane proteins not defined"]},{"year":2015,"claim":"Defined how AMPH-1 enables cargo exit from early endosomes, linking its membrane role to Rab GTPase regulation.","evidence":"Co-IP/pulldown, genetic loss-of-function, in vivo colocalization, and epistasis showing AMPH-1/RAB-10 recruit the RAB-5 GAP TBC-2 in C. elegans","pmids":["26393361"],"confidence":"High","gaps":["Structural details of the AMPH-1–TBC-2–RAB-10 complex unknown","Whether membrane fission and RAB-5 downregulation are mechanistically coupled not established"]},{"year":2022,"claim":"Showed AMPH-1 alone is sufficient for membrane fission and that GTP unexpectedly stimulates this activity, redefining N-BAR proteins as fission catalysts rather than passive tubulators.","evidence":"In vitro fission assay (burst analysis spectroscopy) with purified protein, H0-helix mutants, GTP addition, and comparison to yeast Rvs161/167p","pmids":["36435193"],"confidence":"High","gaps":["Mechanism by which GTP acts on a non-canonical GTPase not defined","Physiological relevance of GTP stimulation in vivo untested"]},{"year":2025,"claim":"Provided the structural mechanism for GTP-regulated tubulation, explaining how nucleotide state switches AMPH-1 between membrane binding and lattice assembly.","evidence":"Structural/biochemical characterization of GTP- vs GDP-bound states, liposome tubulation, and N-terminal helix mutagenesis","pmids":["40749062"],"confidence":"High","gaps":["High-resolution structure of the assembled lattice not reported","How fission is triggered downstream of lattice formation unresolved"]},{"year":2026,"claim":"Linked AMPH tubulation to T-tubule biogenesis and revealed regulation by linear ubiquitination, adding a post-translational control layer to membrane remodeling.","evidence":"Drosophila LUBEL loss-of-function, direct Amph–LUBEL interaction assay, T-tubule morphology imaging, and ubiquitin ligase activity assays","pmids":["41499502"],"confidence":"Medium","gaps":["Ubiquitination sites on Amph and their functional consequence not mapped","Conservation claimed but not tested in mammalian muscle"]},{"year":2018,"claim":"Proposed AMPH as a negative regulator of Ras-Raf-MEK-ERK (and EMT) signaling in cancer cells, raising a possible non-membrane-trafficking role.","evidence":"shRNA knockdown/overexpression in lung and breast cancer cell lines with pathway western blots, proliferation/migration assays, and xenografts","pmids":["30143925","29937937"],"confidence":"Low","gaps":["Pathway placement is correlative; no direct molecular interaction with ERK pathway components demonstrated","Mechanistic link between membrane remodeling and ERK regulation unknown","Single-lab perturbation approach without reconstitution"]},{"year":null,"claim":"How the membrane-remodeling and Rab-regulatory functions of AMPH are integrated in mammalian tissues, and whether its reported signaling role is mechanistically connected to its trafficking activity, remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No mammalian reconstitution connecting fission, RAB-5 downregulation, and ERK regulation","Substrate/cargo specificity in human cells undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[2,3]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[1]}],"localization":[{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[0,1]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[5]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[4]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[0,2]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[0,1]}],"complexes":[],"partners":["RME-1/EHD1","TBC-2","RAB-10","LUBEL/RNF31"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P49418","full_name":"Amphiphysin","aliases":[],"length_aa":695,"mass_kda":76.3,"function":"May participate in mechanisms of regulated exocytosis in synapses and certain endocrine cell types. May control the properties of the membrane associated cytoskeleton","subcellular_location":"Cytoplasmic vesicle, secretory vesicle, synaptic vesicle membrane; Cytoplasm, cytoskeleton","url":"https://www.uniprot.org/uniprotkb/P49418/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/AMPH","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"BIN1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/AMPH","total_profiled":1310},"omim":[{"mim_id":"620664","title":"RHO GUANINE NUCLEOTIDE EXCHANGE FACTOR 37; ARHGEF37","url":"https://www.omim.org/entry/620664"},{"mim_id":"609410","title":"SYNAPTOJANIN 2; SYNJ2","url":"https://www.omim.org/entry/609410"},{"mim_id":"606396","title":"BRIDGING INTEGRATOR 3; BIN3","url":"https://www.omim.org/entry/606396"},{"mim_id":"605936","title":"BRIDGING INTEGRATOR 2; BIN2","url":"https://www.omim.org/entry/605936"},{"mim_id":"604960","title":"PROTEIN KINASE C AND CASEIN KINASE SUBSTRATE IN NEURONS 2; PACSIN2","url":"https://www.omim.org/entry/604960"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"},{"location":"Plasma membrane","reliability":"Additional"}],"tissue_specificity":"Group enriched","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"brain","ntpm":51.0},{"tissue":"pituitary gland","ntpm":20.1},{"tissue":"retina","ntpm":61.1}],"url":"https://www.proteinatlas.org/search/AMPH"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"P49418","domains":[{"cath_id":"1.20.1270.60","chopping":"8-238","consensus_level":"high","plddt":94.2754,"start":8,"end":238},{"cath_id":"2.30.30.40","chopping":"626-695","consensus_level":"high","plddt":91.816,"start":626,"end":695}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P49418","model_url":"https://alphafold.ebi.ac.uk/files/AF-P49418-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P49418-F1-predicted_aligned_error_v6.png","plddt_mean":63.12},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=AMPH","jax_strain_url":"https://www.jax.org/strain/search?query=AMPH"},"sequence":{"accession":"P49418","fasta_url":"https://rest.uniprot.org/uniprotkb/P49418.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P49418/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P49418"}},"corpus_meta":[{"pmid":"19915558","id":"PMC_19915558","title":"AMPH-1/Amphiphysin/Bin1 functions with RME-1/Ehd1 in endocytic recycling.","date":"2009","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/19915558","citation_count":168,"is_preprint":false},{"pmid":"9324260","id":"PMC_9324260","title":"AmpC and AmpH, proteins related to the class C beta-lactamases, bind penicillin and contribute to the normal morphology of Escherichia coli.","date":"1997","source":"Journal of bacteriology","url":"https://pubmed.ncbi.nlm.nih.gov/9324260","citation_count":83,"is_preprint":false},{"pmid":"22001512","id":"PMC_22001512","title":"AmpH, a bifunctional DD-endopeptidase and DD-carboxypeptidase of Escherichia coli.","date":"2011","source":"Journal of bacteriology","url":"https://pubmed.ncbi.nlm.nih.gov/22001512","citation_count":51,"is_preprint":false},{"pmid":"25279949","id":"PMC_25279949","title":"Characterization of the Neurospora crassa cell fusion proteins, HAM-6, HAM-7, HAM-8, HAM-9, HAM-10, AMPH-1 and WHI-2.","date":"2014","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/25279949","citation_count":39,"is_preprint":false},{"pmid":"11062187","id":"PMC_11062187","title":"Aeromonas hydrophila AmpH and CepH beta-lactamases: derepressed expression in mutants of Escherichia coli lacking creB.","date":"2000","source":"The Journal of antimicrobial chemotherapy","url":"https://pubmed.ncbi.nlm.nih.gov/11062187","citation_count":35,"is_preprint":false},{"pmid":"26393361","id":"PMC_26393361","title":"Basolateral Endocytic Recycling Requires RAB-10 and AMPH-1 Mediated Recruitment of RAB-5 GAP TBC-2 to Endosomes.","date":"2015","source":"PLoS genetics","url":"https://pubmed.ncbi.nlm.nih.gov/26393361","citation_count":32,"is_preprint":false},{"pmid":"7757077","id":"PMC_7757077","title":"Primary structure of human amphiphysin, the dominant autoantigen of paraneoplastic stiff-man syndrome, and mapping of its gene (AMPH) to chromosome 7p13-p14.","date":"1995","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/7757077","citation_count":28,"is_preprint":false},{"pmid":"10668437","id":"PMC_10668437","title":"Changes in mRNA levels for heat-shock/stress proteins (Hsp) and a secretory vesicle associated cysteine-string protein (Csp1) after amphetamine (AMPH) exposure.","date":"1999","source":"Annals of the New York Academy of Sciences","url":"https://pubmed.ncbi.nlm.nih.gov/10668437","citation_count":18,"is_preprint":false},{"pmid":"31685299","id":"PMC_31685299","title":"miR-425 regulates cell proliferation, migration and apoptosis by targeting AMPH-1 in non-small-cell lung cancer.","date":"2019","source":"Pathology, research and practice","url":"https://pubmed.ncbi.nlm.nih.gov/31685299","citation_count":16,"is_preprint":false},{"pmid":"30143925","id":"PMC_30143925","title":"AMPH-1 is a tumor suppressor of lung cancer by inhibiting Ras-Raf-MEK-ERK signal pathway.","date":"2018","source":"Lasers in medical 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membrane fission by the N-BAR protein AMPH-1.","date":"2022","source":"Traffic (Copenhagen, Denmark)","url":"https://pubmed.ncbi.nlm.nih.gov/36435193","citation_count":2,"is_preprint":false},{"pmid":"35398334","id":"PMC_35398334","title":"Impact of two different types of exercise training on AMPH addiction: Role of hippocampal neurotrophins.","date":"2022","source":"Physiology & behavior","url":"https://pubmed.ncbi.nlm.nih.gov/35398334","citation_count":2,"is_preprint":false},{"pmid":"41544047","id":"PMC_41544047","title":"Machine learning and network pharmacology identify keloid biomarkers (AMPH, TNFRSF9) and therapeutic targets (IL6, HAS2) for aloe-derived quercetin.","date":"2026","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/41544047","citation_count":1,"is_preprint":false},{"pmid":"40749062","id":"PMC_40749062","title":"GTP hydrolysis triggers membrane remodeling by AMPH-1.","date":"2025","source":"Science 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membrane tubules distinct from those produced by either protein alone, indicating cooperative regulation of endocytic recycling.\",\n      \"method\": \"In vivo colocalization (live imaging), deletion mutant phenotypic analysis, in vitro reconstitution of membrane tubulation with purified proteins, EH-domain binding assay\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro reconstitution with purified proteins, genetic loss-of-function with defined cellular phenotype, direct binding assay, and functional rescue; multiple orthogonal methods in one study\",\n      \"pmids\": [\"19915558\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"RAB-10/Rab10 and AMPH-1/Amphiphysin directly bind the RAB-5 GAP TBC-2 and recruit it to endosomes; in the absence of RAB-10 or AMPH-1 binding to TBC-2, RAB-5 membrane association is abnormally elevated and recycling cargo is trapped in early endosomes, establishing that AMPH-1–mediated downregulation of RAB-5 is required for cargo exit from early endosomes.\",\n      \"method\": \"Binding assays (Co-IP/pulldown), genetic loss-of-function (deletion mutants), fluorescence colocalization in vivo, epistasis analysis\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal binding demonstrated, genetic epistasis with defined cargo-recycling phenotype, multiple orthogonal methods in one study\",\n      \"pmids\": [\"26393361\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Purified C. elegans AMPH-1 alone is sufficient to drive membrane fission, converting large unilamellar vesicles into small tubular-vesicular products; this fission requires the amphipathic H0 helix of AMPH-1; RME-1 slows fission; unexpectedly, GTP stimulates AMPH-1-induced membrane fission. The yeast heterodimeric N-BAR protein Rvs161/167p shows the same GTP-stimulated fission, suggesting this is a general property of N-BAR proteins.\",\n      \"method\": \"In vitro membrane fission assay using burst analysis spectroscopy (BAS) with purified protein, H0-helix mutant analysis, GTP addition experiment, comparative assay with yeast Rvs161/167p\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstitution with purified protein, mutagenesis of H0 helix, multiple conditions tested in one study; single lab but orthogonal methods\",\n      \"pmids\": [\"36435193\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"GTP binding stabilizes interactions between AMPH-1 and the membrane through amphipathic N-terminal α-helices at the tips of the arc-shaped homodimeric structure; GDP-bound (post-hydrolysis) AMPH-1 repositions these helices to interact with helices of other homodimers, forming an oligomeric AMPH-1 lattice that tubulates membranes in preparation for carrier formation by membrane fission.\",\n      \"method\": \"Structural and biochemical characterization of GTP- vs. GDP-bound states, liposome tubulation assays, mutational analysis of N-terminal helices\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — structural model linked to functional membrane-tubulation assay with mutagenesis, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"40749062\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"In Drosophila muscle, LUBEL/RNF31 (a ubiquitin E3 ligase for linear/M1-linked ubiquitination) directly interacts with Amphiphysin (Amph, a BAR-domain protein); LUBEL ubiquitin ligase activity and the LUBEL–Amph interaction are required for proper T-tubule morphology; loss of LUBEL produces Amph-positive membrane sheets instead of tubular networks; LUBEL and M1-linked ubiquitin chains assemble into puncta on membranes through multivalent interactions, facilitating Amph-mediated membrane tubulation. The Amph–LUBEL/RNF31 interaction is evolutionarily conserved.\",\n      \"method\": \"Genetic loss-of-function (LUBEL mutants in Drosophila), Co-IP/direct interaction assay, fluorescence microscopy of T-tubule morphology, ubiquitin ligase activity assays\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal interaction demonstrated, genetic loss-of-function with defined morphological phenotype, multiple methods; Drosophila ortholog study, conserved interaction noted but single lab\",\n      \"pmids\": [\"41499502\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"Human amphiphysin (AMPH) is peripherally associated with synaptic vesicles; it is expressed in neurons, certain endocrine cell types, and spermatocytes; the gene was mapped to chromosome 7p13-p14; autoantibodies against amphiphysin occur in paraneoplastic Stiff-Man syndrome.\",\n      \"method\": \"cDNA cloning, primary structure determination, chromosomal mapping, immunolocalization\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct subcellular localization by biochemical fractionation (synaptic vesicle association), cDNA-level primary structure, replicated in human and chicken; no functional mutagenesis\",\n      \"pmids\": [\"7757077\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Knockdown of AMPH-1 in lung cancer cells activates the Ras-Raf-MEK-ERK signaling pathway, promoting cell proliferation, attenuating apoptosis, and accelerating cell cycle progression; overexpression reverses these effects, placing AMPH-1 as a negative regulator of this pathway.\",\n      \"method\": \"shRNA knockdown and overexpression in lung cancer cell lines, western blot for ERK pathway components, in vivo xenograft mouse model\",\n      \"journal\": \"Lasers in medical science\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single KD/OE approach with pathway readout but no direct binding or reconstitution demonstrating mechanism; pathway placement is correlative\",\n      \"pmids\": [\"30143925\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Knockdown of AMPH-1 in breast cancer cells activates ERK and EMT pathways, promoting proliferation, migration, and cell cycle progression while attenuating apoptosis, consistent with AMPH-1 acting as a negative regulator of ERK and EMT signaling.\",\n      \"method\": \"shRNA knockdown in breast cancer cell lines, western blot for ERK/EMT markers, proliferation/migration assays, in vivo xenograft\",\n      \"journal\": \"Journal of Cancer\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, pathway placement by KD with no direct molecular interaction demonstrated\",\n      \"pmids\": [\"29937937\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"AMPH/Amphiphysin is a BAR-domain (N-BAR) protein that uses its amphipathic H0 helix to sense and deform membranes; GTP binding stabilizes its membrane interaction through these N-terminal helices, while GTP hydrolysis drives repositioning of the helices to form an oligomeric lattice that tubulates membranes, ultimately enabling membrane fission to produce transport carriers from the recycling endosome; it cooperates with RME-1/EHD1 (binding via NPF sequences to the EH-domain) to recycle transmembrane cargo, and promotes endosomal maturation by recruiting the RAB-5 GAP TBC-2 together with RAB-10 to downregulate RAB-5 and allow cargo exit from early endosomes; in muscle, its membrane-tubulating activity is regulated by linear ubiquitination via the LUBEL/RNF31 E3 ligase, which is required for T-tubule biogenesis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"AMPH/Amphiphysin is an N-BAR-domain protein that senses and remodels membranes to generate transport carriers during endocytic recycling and membrane-tubule biogenesis [#0, #2]. Purified AMPH-1 is sufficient to deform membranes and drive fission, converting vesicles into tubular-vesicular products in a reaction that depends on its amphipathic H0 helix and is stimulated by GTP [#2]. GTP binding stabilizes membrane contacts through the N-terminal helices at the tips of the arc-shaped homodimer, and post-hydrolysis (GDP-bound) repositioning of these helices drives assembly of an oligomeric AMPH-1 lattice that tubulates the membrane prior to fission [#3]. In endocytic recycling, AMPH-1 colocalizes with and binds the EH-domain of RME-1/EHD1 through its NPF/D/E motifs, and the two proteins cooperatively produce coated membrane tubules and are required for recycling-endosome morphology and cargo recycling [#0]; AMPH-1 further promotes cargo exit from early endosomes by binding RAB-10 and recruiting the RAB-5 GAP TBC-2 to downregulate RAB-5 [#1]. In muscle, AMPH-mediated tubulation underlies T-tubule biogenesis and is controlled by linear (M1-linked) ubiquitination through direct interaction with the E3 ligase LUBEL/RNF31, whose activity is required to convert Amph-positive membrane sheets into tubular networks [#4]. AMPH was originally identified as a synaptic-vesicle-associated neuronal protein and the autoantigen in paraneoplastic Stiff-Man syndrome [#5].\",\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"Established that AMPH-1 acts in endocytic recycling by partnering with RME-1/EHD1, answering whether this BAR protein functions beyond synaptic endocytosis.\",\n      \"evidence\": \"In vivo colocalization, amph-1 deletion phenotypes, EH-domain binding assay, and in vitro tubulation with purified AMPH-1\\u2013RME-1 in C. elegans\",\n      \"pmids\": [\"19915558\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the structural basis of tubulation\", \"Cargo specificity and the full repertoire of recycled transmembrane proteins not defined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined how AMPH-1 enables cargo exit from early endosomes, linking its membrane role to Rab GTPase regulation.\",\n      \"evidence\": \"Co-IP/pulldown, genetic loss-of-function, in vivo colocalization, and epistasis showing AMPH-1/RAB-10 recruit the RAB-5 GAP TBC-2 in C. elegans\",\n      \"pmids\": [\"26393361\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural details of the AMPH-1\\u2013TBC-2\\u2013RAB-10 complex unknown\", \"Whether membrane fission and RAB-5 downregulation are mechanistically coupled not established\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Showed AMPH-1 alone is sufficient for membrane fission and that GTP unexpectedly stimulates this activity, redefining N-BAR proteins as fission catalysts rather than passive tubulators.\",\n      \"evidence\": \"In vitro fission assay (burst analysis spectroscopy) with purified protein, H0-helix mutants, GTP addition, and comparison to yeast Rvs161/167p\",\n      \"pmids\": [\"36435193\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which GTP acts on a non-canonical GTPase not defined\", \"Physiological relevance of GTP stimulation in vivo untested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Provided the structural mechanism for GTP-regulated tubulation, explaining how nucleotide state switches AMPH-1 between membrane binding and lattice assembly.\",\n      \"evidence\": \"Structural/biochemical characterization of GTP- vs GDP-bound states, liposome tubulation, and N-terminal helix mutagenesis\",\n      \"pmids\": [\"40749062\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"High-resolution structure of the assembled lattice not reported\", \"How fission is triggered downstream of lattice formation unresolved\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Linked AMPH tubulation to T-tubule biogenesis and revealed regulation by linear ubiquitination, adding a post-translational control layer to membrane remodeling.\",\n      \"evidence\": \"Drosophila LUBEL loss-of-function, direct Amph\\u2013LUBEL interaction assay, T-tubule morphology imaging, and ubiquitin ligase activity assays\",\n      \"pmids\": [\"41499502\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ubiquitination sites on Amph and their functional consequence not mapped\", \"Conservation claimed but not tested in mammalian muscle\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Proposed AMPH as a negative regulator of Ras-Raf-MEK-ERK (and EMT) signaling in cancer cells, raising a possible non-membrane-trafficking role.\",\n      \"evidence\": \"shRNA knockdown/overexpression in lung and breast cancer cell lines with pathway western blots, proliferation/migration assays, and xenografts\",\n      \"pmids\": [\"30143925\", \"29937937\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Pathway placement is correlative; no direct molecular interaction with ERK pathway components demonstrated\", \"Mechanistic link between membrane remodeling and ERK regulation unknown\", \"Single-lab perturbation approach without reconstitution\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the membrane-remodeling and Rab-regulatory functions of AMPH are integrated in mammalian tissues, and whether its reported signaling role is mechanistically connected to its trafficking activity, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No mammalian reconstitution connecting fission, RAB-5 downregulation, and ERK regulation\", \"Substrate/cargo specificity in human cells undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [0, 2]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"RME-1/EHD1\", \"TBC-2\", \"RAB-10\", \"LUBEL/RNF31\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}