Affinage

AMPH

Amphiphysin · UniProt P49418

Length
695 aa
Mass
76.3 kDa
Annotated
2026-06-09
19 papers in source corpus 8 papers cited in narrative 8 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 6/6 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

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).

Mechanistic history

Synthesis pass · year-by-year structured walk · 6 steps
  1. 2009 High

    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

    PMID:19915558

    Open questions at the time
    • Did not resolve the structural basis of tubulation
    • Cargo specificity and the full repertoire of recycled transmembrane proteins not defined
  2. 2015 High

    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

    PMID:26393361

    Open questions at the time
    • Structural details of the AMPH-1–TBC-2–RAB-10 complex unknown
    • Whether membrane fission and RAB-5 downregulation are mechanistically coupled not established
  3. 2022 High

    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

    PMID:36435193

    Open questions at the time
    • Mechanism by which GTP acts on a non-canonical GTPase not defined
    • Physiological relevance of GTP stimulation in vivo untested
  4. 2025 High

    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

    PMID:40749062

    Open questions at the time
    • High-resolution structure of the assembled lattice not reported
    • How fission is triggered downstream of lattice formation unresolved
  5. 2026 Medium

    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

    PMID:41499502

    Open questions at the time
    • Ubiquitination sites on Amph and their functional consequence not mapped
    • Conservation claimed but not tested in mammalian muscle
  6. 2018 Low

    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

    PMID:29937937 PMID:30143925

    Open questions at the time
    • 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

Open questions

Synthesis pass · forward-looking unresolved questions
  • 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.
  • No mammalian reconstitution connecting fission, RAB-5 downregulation, and ERK regulation
  • Substrate/cargo specificity in human cells undefined

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0008289 lipid binding 2 GO:0098772 molecular function regulator activity 1
Localization
GO:0005768 endosome 2 GO:0005886 plasma membrane 1 GO:0031410 cytoplasmic vesicle 1
Pathway
R-HSA-5653656 Vesicle-mediated transport 2 R-HSA-9609507 Protein localization 2
Partners
LUBEL/RNF31RAB-10RME-1/EHD1TBC-2

Evidence

Reading pass · 8 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2009 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. In vivo colocalization (live imaging), deletion mutant phenotypic analysis, in vitro reconstitution of membrane tubulation with purified proteins, EH-domain binding assay Nature cell biology High 19915558
2015 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. Binding assays (Co-IP/pulldown), genetic loss-of-function (deletion mutants), fluorescence colocalization in vivo, epistasis analysis PLoS genetics High 26393361
2022 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. 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 Traffic (Copenhagen, Denmark) High 36435193
2025 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. Structural and biochemical characterization of GTP- vs. GDP-bound states, liposome tubulation assays, mutational analysis of N-terminal helices Science advances High 40749062
2026 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. Genetic loss-of-function (LUBEL mutants in Drosophila), Co-IP/direct interaction assay, fluorescence microscopy of T-tubule morphology, ubiquitin ligase activity assays Science advances Medium 41499502
1995 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. cDNA cloning, primary structure determination, chromosomal mapping, immunolocalization Human molecular genetics Medium 7757077
2018 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. shRNA knockdown and overexpression in lung cancer cell lines, western blot for ERK pathway components, in vivo xenograft mouse model Lasers in medical science Low 30143925
2018 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. shRNA knockdown in breast cancer cell lines, western blot for ERK/EMT markers, proliferation/migration assays, in vivo xenograft Journal of Cancer Low 29937937

Source papers

Stage 0 corpus · 19 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2009 AMPH-1/Amphiphysin/Bin1 functions with RME-1/Ehd1 in endocytic recycling. Nature cell biology 168 19915558
1997 AmpC and AmpH, proteins related to the class C beta-lactamases, bind penicillin and contribute to the normal morphology of Escherichia coli. Journal of bacteriology 83 9324260
2011 AmpH, a bifunctional DD-endopeptidase and DD-carboxypeptidase of Escherichia coli. Journal of bacteriology 51 22001512
2014 Characterization of the Neurospora crassa cell fusion proteins, HAM-6, HAM-7, HAM-8, HAM-9, HAM-10, AMPH-1 and WHI-2. PloS one 39 25279949
2000 Aeromonas hydrophila AmpH and CepH beta-lactamases: derepressed expression in mutants of Escherichia coli lacking creB. The Journal of antimicrobial chemotherapy 35 11062187
2015 Basolateral Endocytic Recycling Requires RAB-10 and AMPH-1 Mediated Recruitment of RAB-5 GAP TBC-2 to Endosomes. PLoS genetics 32 26393361
1995 Primary structure of human amphiphysin, the dominant autoantigen of paraneoplastic stiff-man syndrome, and mapping of its gene (AMPH) to chromosome 7p13-p14. Human molecular genetics 28 7757077
1999 Changes in mRNA levels for heat-shock/stress proteins (Hsp) and a secretory vesicle associated cysteine-string protein (Csp1) after amphetamine (AMPH) exposure. Annals of the New York Academy of Sciences 18 10668437
2019 miR-425 regulates cell proliferation, migration and apoptosis by targeting AMPH-1 in non-small-cell lung cancer. Pathology, research and practice 16 31685299
2018 AMPH-1 is a tumor suppressor of lung cancer by inhibiting Ras-Raf-MEK-ERK signal pathway. Lasers in medical science 10 30143925
2018 AMPH-1 is critical for breast cancer progression. Journal of Cancer 9 29937937
2006 Identification of informative strains and provisional QTL mapping of amphetamine (AMPH)-induced locomotion in recombinant congenic strains (RCS) of mice. Behavior genetics 5 16710777
2019 AMPH-1 As A Critical Tumor Suppressor That Inhibits Osteosarcoma Progression. Cancer management and research 3 31819629
2022 Impact of two different types of exercise training on AMPH addiction: Role of hippocampal neurotrophins. Physiology & behavior 2 35398334
2022 GTP-stimulated membrane fission by the N-BAR protein AMPH-1. Traffic (Copenhagen, Denmark) 2 36435193
2026 Machine learning and network pharmacology identify keloid biomarkers (AMPH, TNFRSF9) and therapeutic targets (IL6, HAS2) for aloe-derived quercetin. PloS one 1 41544047
2026 Linear ubiquitination triggers Amph-mediated T-tubule biogenesis. Science advances 0 41499502
2025 GTP hydrolysis triggers membrane remodeling by AMPH-1. Science advances 0 40749062
2025 Regulatory relationships among aldB, ampH, and acoR and their impact on β-lactam susceptibility in Phytobacter diazotrophicus. Frontiers in microbiology 0 41311497

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