{"gene":"PDCD6IP","run_date":"2026-04-29T11:37:58","timeline":{"discoveries":[{"year":1999,"finding":"ALIX/AIP1 (PDCD6IP) was identified as a novel protein that interacts with the calcium-binding protein ALG-2 in a Ca2+-dependent manner; the interaction requires calcium as an indispensable requisite, and both proteins colocalize in the cytosol. Overexpression of a truncated AIP1 protects cells from trophic factor withdrawal-induced death.","method":"Yeast two-hybrid screening, colocalization, overexpression assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — original identification with yeast two-hybrid and functional overexpression data, single lab","pmids":["9880530"],"is_preprint":false},{"year":2003,"finding":"ALIX/AIP1 functions in HIV-1 and EIAV retrovirus budding by binding to a secondary late-domain (L-domain) region in HIV-1 Gag p6 and to the EIAV p9 L-domain; ALIX also interacts with Tsg101 and ESCRT-III homologs, serving to link viral Gag to the host ESCRT-III budding machinery.","method":"Co-immunoprecipitation, binding assays, virus budding assays","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, functional budding assays, replicated across retroviruses, highly cited","pmids":["14505569"],"is_preprint":false},{"year":2003,"finding":"ALIX interacts directly with CHMP4b (a yeast Snf7 homolog and ESCRT-III component) via its N-terminal Bro1 domain, and this interaction was confirmed by GST pulldown and co-immunoprecipitation; both proteins colocalize at perinuclear endosomal structures and cooperate in multivesicular body (MVB) formation.","method":"Yeast two-hybrid, GST pulldown, co-immunoprecipitation, fluorescence microscopy","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (pulldown + Co-IP + localization), independently confirmed","pmids":["12860994"],"is_preprint":false},{"year":2004,"finding":"The ALG-2-binding site on ALIX maps to amino acid residues 794–827 in the C-terminal proline-rich region, encompassing four PxY repeats; Ca2+-bound ALG-2 interaction with ALIX is required for the punctate subcellular distribution of the ALIX C-terminal fragment.","method":"Yeast two-hybrid, co-immunoprecipitation, alanine-scanning mutagenesis, fluorescence microscopy","journal":"Journal of biochemistry","confidence":"High","confidence_rationale":"Tier 1 — mutagenesis mapped binding site, orthogonal Co-IP and localization assays","pmids":["14999017"],"is_preprint":false},{"year":2004,"finding":"ALIX/AIP1 inhibits EGFR internalization and downregulation by antagonizing the Cbl–CIN85/SETA complex; ALIX reduces Cbl-mediated ubiquitination and the interaction between Cbl and CIN85, while siRNA-mediated knockdown of Alix promotes EGFR internalization and degradation.","method":"Co-immunoprecipitation, siRNA knockdown, receptor internalization and ubiquitination assays","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP plus loss-of-function with specific phenotypic readout, single lab","pmids":["15456872"],"is_preprint":false},{"year":2004,"finding":"Alix binds to the transferrin receptor (TfR) cytosolic YTRF motif and contributes to sorting of TfR into exosomes during reticulocyte maturation; AP2 degradation via the proteasome relieves competition at this site and allows hsc70 and Alix to drive TfR exosomal sorting.","method":"Co-immunoprecipitation, peptide affinity chromatography, proteasome inhibitor experiments","journal":"Traffic (Copenhagen, Denmark)","confidence":"Medium","confidence_rationale":"Tier 3 — Co-IP and pulldown, single lab with functional inhibitor data","pmids":["15086793"],"is_preprint":false},{"year":2004,"finding":"Paraptosis (a nonapoptotic cell death program induced by the IGF-I receptor) is specifically inhibited by AIP1/Alix, and this inhibition is distinct from apoptosis inhibition; the inhibition does not involve caspase pathways and is mediated via MAP kinases.","method":"Overexpression and dominant-negative AIP1, cell death assays, caspase activity assays","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 — clean overexpression/dominant-negative with specific phenotypic readout, single lab","pmids":["15195070"],"is_preprint":false},{"year":2004,"finding":"Src kinase phosphorylates ALIX at its C-terminal tyrosine-rich region (via an SH3-SH2 interaction with ALIX), which causes ALIX to translocate from membrane/cytoskeleton to the cytoplasm and disrupts ALIX interactions with CIN85/SETA, EGFR, and Pyk2, antagonizing ALIX's inhibitory role in receptor internalization.","method":"In vitro kinase assays, co-immunoprecipitation, subcellular fractionation, overexpression/mutagenesis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1–2 — in vitro kinase assay plus functional co-IP and localization data, single lab","pmids":["15557335"],"is_preprint":false},{"year":2005,"finding":"Depletion of Alix by RNAi causes redistribution of early endosomes from peripheral to perinuclear location and accumulation of unusual cortactin-containing actin structures, indicating Alix functions in actin-dependent intracellular positioning of endosomes; lysobisphosphatidic acid (LBPA) levels in MVEs are reduced upon Alix depletion.","method":"RNAi knockdown, immunofluorescence, immunoelectron microscopy","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 — RNAi knockdown with specific morphological phenotype and quantitative EM, single lab","pmids":["15914539"],"is_preprint":false},{"year":2006,"finding":"ALIX directly binds filamentous actin (F-actin) through both its N-terminal Bro1 domain and C-terminal proline-rich domain; in cells, ALIX also binds cortactin (via the N-terminal half of the V domain) and α-actinin (via the C-terminal half of the V domain), and is required for lamellipodial cortactin localization and stress fiber assembly.","method":"Cell-free actin-binding assays, co-immunoprecipitation, siRNA knockdown, immunofluorescence","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — in vitro binding plus Co-IP and RNAi functional assays, single lab","pmids":["16966331"],"is_preprint":false},{"year":2007,"finding":"Crystal structures of human ALIX reveal it is composed of an N-terminal Bro1 domain and a central V domain (two three-helix bundles forming elongated arms). YPX(n)L late-domain motifs of retroviruses bind a conserved hydrophobic pocket on the second arm of the V domain; CHMP4/ESCRT-III proteins bind a conserved hydrophobic patch on the Bro1 domain; both interactions are required for virus budding.","method":"X-ray crystallography, mutagenesis, virus budding assays","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 — crystal structures with mutagenesis validation and functional budding assay","pmids":["17350572"],"is_preprint":false},{"year":2007,"finding":"The structure of the ALIX V domain bound to HIV-1 p6 LYPXnL motif was solved; Phe676 in a large hydrophobic pocket is crucial for binding; overexpression of the V domain alone inhibits HIV-1 release, and this is reversed by mutations blocking V domain–p6 interaction.","method":"X-ray crystallography, mutagenesis, virus budding inhibition assay","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with site-specific mutagenesis and functional rescue","pmids":["17277784"],"is_preprint":false},{"year":2007,"finding":"Crystal structures of ALIX V domain in complex with YPX(n)L late domains from HIV-1 and EIAV show that distinct late domains bind the same hydrophobic site on the V domain adopting different conformations; binding affinities are context-dependent and key interface residues were validated functionally.","method":"X-ray crystallography, surface plasmon resonance, mutagenesis, virus budding assays","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 — dual crystal structures with biophysical binding measurements and functional assays","pmids":["18066081"],"is_preprint":false},{"year":2007,"finding":"ALIX is recruited to the midbody of dividing cells through direct binding between the central CEP55 'hinge' region and a GPP-based motif in ALIX; depletion of ALIX (and TSG101) inhibits the abscission step of cytokinesis; ALIX point mutants blocking CEP55 and CHMP4/ESCRT-III binding also block abscission.","method":"Proteomics, co-immunoprecipitation, RNAi knockdown, dominant-negative mutants, live cell imaging","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods, replicated across studies, highly cited","pmids":["17853893"],"is_preprint":false},{"year":2007,"finding":"ALIX potently rescues HIV-1 L-domain mutant budding; this rescue depends on intact ALIX–p6 binding site and the ESCRT-III (CHMP4B)-binding surface on the Bro1 domain; ALIX C-terminal proline-rich domain (PRD) is also required but independently of TSG101/endophilin/CIN85 binding sites.","method":"Mutagenesis, virus budding assays, co-immunoprecipitation","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 2 — systematic mutagenesis with functional budding readouts","pmids":["17428861"],"is_preprint":false},{"year":2007,"finding":"ALIX is recruited to the cytokinetic midbody via Cep55 interaction through an evolutionarily conserved peptide; disruption of Cep55/Alix/ESCRT-III interactions causes aberrant midbodies and cytokinetic failure; ALIX C-terminus encodes a multimerization activity essential for Alix-dependent HIV-1 release and Tsg101 interaction.","method":"Co-immunoprecipitation, dominant-negative mutants, siRNA knockdown, cytokinesis failure assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal assays establishing mechanistic links in both cytokinesis and virus budding","pmids":["18641129"],"is_preprint":false},{"year":2007,"finding":"HIV-1 ALIX also engages the nucleocapsid (NC) domain of Gag via its Bro1 domain (zinc finger-dependent, RNA-independent) allowing ALIX incorporation into virions independently of the p6 L-domain; NC zinc finger mutations reduce particle production and show functional relationship with the p6 PTAP motif.","method":"Co-immunoprecipitation, nuclease treatment, HIV budding assays, ALIX overexpression rescue","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP with controls plus functional budding assays, single lab","pmids":["18032513"],"is_preprint":false},{"year":2008,"finding":"Crystal structures of the ALIX Bro1 domain bound to C-terminal peptides of CHMP4A-C reveal that CHMP4 C-terminal amphipathic helices bind across the concave surface of Bro1; key hydrophobic residues (M/L/IxxLxxW pattern) mediate binding; mutations in exposed Bro1 surface residues block ALIX-dependent HIV-1 budding.","method":"X-ray crystallography, mutagenesis, HIV-1 budding assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — crystal structures with mutagenesis and functional validation","pmids":["18511562"],"is_preprint":false},{"year":2008,"finding":"ALG-2 crystal structure in complex with the Alix 799-814 peptide reveals a Ca2+/EF3-driven arginine switch: Ca2+ binding to EF3 moves Arg125 to open a hydrophobic pocket that accommodates the PPYP motif of Alix, explaining the Ca2+-dependence of ALG-2/Alix association.","method":"X-ray crystallography, in vitro binding assays with mutant proteins","journal":"Structure (London, England : 1993)","confidence":"High","confidence_rationale":"Tier 1 — crystal structures with mutagenesis validation of binding mechanism","pmids":["18940611"],"is_preprint":false},{"year":2008,"finding":"AIP1 (DAB2IP) interacts with IRE1 via its PH domain and facilitates IRE1 dimerization, a critical step for IRE1 signaling; AIP1-deficient cells and mice show selectively impaired ER stress-induced IRE1-JNK/XBP-1 signaling but normal PERK-CHOP signaling.","method":"AIP1 knockout mice, co-immunoprecipitation, reconstitution with deletion mutants, in vivo signaling assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — KO mice plus domain-mapping co-IP and reconstitution, specific axis validated","pmids":["18281285"],"is_preprint":false},{"year":2009,"finding":"ALIX dimerizes via its V domain in solution; SAXS modeling reveals a crescent-shaped dimeric conformation; dimeric ALIX colocalizes with and bridges CHMP4B filaments; dimerization-disrupting mutations impair HIV-1 budding, indicating dimeric ALIX is the active form that interacts with ESCRT-III CHMP4 polymers.","method":"SAXS, mutagenesis, co-localization, HIV-1 budding assay, in vitro CHMP4B filament bridging","journal":"Structure (London, England : 1993)","confidence":"High","confidence_rationale":"Tier 1 — structural analysis (SAXS) with mutagenesis and functional budding assays","pmids":["19523902"],"is_preprint":false},{"year":2009,"finding":"ALG-2 acts as a Ca2+-dependent adaptor that bridges Alix and TSG101; the ALG-2-binding site on Alix (not the PSAP/TSG101-UEV binding site) is required for Ca2+-dependent TSG101 pulldown; ALG-2 dimerization and Ca2+ binding are both required for bridging.","method":"Strep-pulldown with mutants, ALG-2 knockdown, recombinant protein reconstitution","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 — domain-mapping pulldown with KD and reconstitution, single lab","pmids":["19520058"],"is_preprint":false},{"year":2010,"finding":"The ALIX C-terminal proline-rich region (PRR) folds back against upstream domains and auto-inhibits V domain binding to viral late domains; mutations that destabilize this closed conformation open the V domain, increase ALIX membrane association, and enhance virus budding.","method":"Mutagenesis, in vitro binding assays, SAXS/structural modeling, virus budding assays","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 1–2 — structural evidence of autoinhibition plus mutagenesis and functional budding assay","pmids":["21715492"],"is_preprint":false},{"year":2010,"finding":"An intramolecular interaction between Patch 2 in the Bro1 domain and the TSG101-docking site in the ALIX PRD locks ALIX in a closed, autoinhibited conformation that prevents CHMP4 and retroviral Gag binding; relieving this intramolecular interaction (by expressing binding partners or deleting interaction sites) enables ALIX to interact with CHMP4 and facilitates membrane association and EIAV budding.","method":"ALIX truncation and mutagenesis, co-immunoprecipitation, EIAV budding assay","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1–2 — systematic domain mapping with functional virus budding readout, consistent with parallel structural data","pmids":["20929444"],"is_preprint":false},{"year":2010,"finding":"SIVmac239 and SIVagmTan-1 possess divergent ALIX-binding late domains distinct from canonical YPX(n)L; crystal structures reveal that anchoring tyrosines and adjacent hydrophobic residues contact the same ALIX V domain hydrophobic pocket used by HIV-1/EIAV late domains.","method":"X-ray crystallography, late-domain mapping, virus budding assays","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 1 — crystal structures defining binding mechanism","pmids":["20962096"],"is_preprint":false},{"year":2011,"finding":"Alix Bro1 domain Surface 1 (concave face, CHMP4 binding site), Surface 2 (narrow end), and a unique extended loop exposing Phe105 all independently contribute to ALIX's ability to stimulate HIV-1 budding, as established by mutagenesis guided by the BROX crystal structure.","method":"X-ray crystallography (BROX), ALIX mutagenesis, HIV-1 budding assays","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 1–2 — structure-guided mutagenesis with functional budding assay, single lab","pmids":["22162750"],"is_preprint":false},{"year":2012,"finding":"ALIX binds the V domain to a YPX(3)L motif in the GPCR PAR1 and mediates ubiquitin-independent ESCRT-III-dependent MVB/lysosomal sorting of PAR1, providing an alternative MVB sorting pathway that bypasses ubiquitin-binding ESCRTs.","method":"Co-immunoprecipitation, RNAi knockdown, mutagenesis of YPX3L motif, MVB sorting assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, site-directed mutagenesis and KD with specific MVB sorting readout","pmids":["22547407"],"is_preprint":false},{"year":2012,"finding":"ALIX is a Lys63-specific polyubiquitin binding protein; the V domain directly and selectively binds K63-linked polyUb chains (preference for chains ≥3 Ub) via two putative Ub-binding sites on a single α-helical surface; mutations in these sites impair polyUb binding and lentivirus budding.","method":"Affinity capture/mass spectrometry, in vitro binding assays, mutagenesis, virus budding assays","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro direct binding with mutagenesis plus functional virus budding assay","pmids":["23201121"],"is_preprint":false},{"year":2012,"finding":"Alix is ubiquitinated by the Ozz-E3 ligase in skeletal muscle; interaction with Ozz changes Alix conformation and promotes its ubiquitination, regulating Alix levels at cortactin-positive actin remodeling subcompartments; Alix knockdown reduces F-actin levels and impairs cell motility in myotubes.","method":"Co-immunoprecipitation, ubiquitination assays, Ozz-/- mouse model, siRNA knockdown","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — KO model plus in-cell ubiquitination and functional actin/motility assays, single lab","pmids":["22334701"],"is_preprint":false},{"year":2012,"finding":"AP-3 adaptor complex binds a tyrosine-based motif in the PAR1 cytoplasmic tail and functions upstream of ALIX to facilitate PAR1 lysosomal sorting; AP-3 promotes PAR1–ALIX interaction, demonstrating a sequential AP-3→ALIX→ESCRT-III pathway for ubiquitin-independent MVB sorting.","method":"Co-immunoprecipitation, RNAi knockdown, mutagenesis, receptor sorting assays","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 — epistasis established by RNAi knockdown with specific receptor-sorting readout, single lab","pmids":["22833563"],"is_preprint":false},{"year":2014,"finding":"The syndecan–syntenin–ALIX pathway controls exosome biogenesis and intraluminal budding; ALIX interaction with syntenin and LBPA is required for ILV budding into MVBs; ARF6 and PLD2 regulate this pathway by controlling ILV budding.","method":"RNAi knockdown, co-immunoprecipitation, nanoparticle tracking, electron microscopy","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods, replicated across subsequent studies","pmids":["24637612"],"is_preprint":false},{"year":2015,"finding":"The ATG12–ATG3 conjugate interacts with Alix (PDCD6IP) and controls multiple Alix-dependent processes including late endosome distribution, exosome biogenesis, and viral budding; cells lacking ATG12–ATG3 phenocopy Alix loss-of-function in terms of basal autophagic flux and perinuclear late endosome accumulation.","method":"Co-immunoprecipitation, KO cell lines, functional assays for autophagy/exosomes/virus budding","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP and loss-of-function in multiple assays across distinct cellular processes, single lab","pmids":["25686249"],"is_preprint":false},{"year":2015,"finding":"Ca2+-dependent ALG-2 interaction with ALIX completely relieves ALIX's intramolecular autoinhibition and promotes CHMP4-dependent ALIX membrane association; EGFR activation increases ALG-2–ALIX interaction and ALIX membrane association; inhibiting ALG-2-mediated ALIX activation blocks MVB sorting of activated EGFR but not cytokinetic abscission or EIAV budding.","method":"Co-immunoprecipitation, mutagenesis, EGFR MVB sorting assays, domain-specific inhibition","journal":"Cell discovery","confidence":"High","confidence_rationale":"Tier 2 — mechanistic domain mapping with context-specific functional readouts, single lab with orthogonal assays","pmids":["27462417"],"is_preprint":false},{"year":2015,"finding":"ALIX mediates MVB sorting and silencing (signal attenuation) of activated ubiquitinated EGFR via V domain interaction with ubiquitinated EGFR and Bro1 domain interaction with membrane-bound CHMP4; inhibiting either ALIX dimerization or CHMP4 interaction blocks EGFR MVB sorting and promotes sustained ERK1/2 activation.","method":"EGF stimulation, co-immunoprecipitation, siRNA knockdown, signaling assays","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 — domain-specific inhibition with functional receptor sorting and signaling readout, single lab","pmids":["25510652"],"is_preprint":false},{"year":2015,"finding":"Heparanase stimulates the syndecan–syntenin–ALIX exosome pathway by trimming heparan sulfate chains on syndecans, fostering endosomal membrane budding; exosomal secretion of syntenin-1, syndecan, and CD63 depends on the syntenin–ALIX interaction.","method":"RNAi knockdown, exosome quantification, co-immunoprecipitation, electron microscopy","journal":"Cell research","confidence":"Medium","confidence_rationale":"Tier 2 — mechanistic epistasis established by syntenin/ALIX KD in heparanase-dependent pathway, single lab","pmids":["25732677"],"is_preprint":false},{"year":2015,"finding":"Nedd4-1 ubiquitin ligase interacts with ALIX (via Co-IP and yeast two-hybrid) and is recruited to facilitate HIV-1 release through the LYPXnL/ALIX pathway; Nedd4-1 catalytic activity ubiquitinates ALIX itself, and RNAi knockdown of Nedd4-1 eliminates ALIX ubiquitination and impairs ALIX-dependent HIV-1 release.","method":"Co-immunoprecipitation, yeast two-hybrid, RNAi knockdown, ubiquitination assay, virus budding assays","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods identifying Nedd4-1 as ALIX writer and functional role in budding, single lab","pmids":["20519395"],"is_preprint":false},{"year":2016,"finding":"ALIX mediates ubiquitin-independent lysosomal sorting of the P2Y1 purinergic GPCR through a YPX3L motif in the receptor's second intracellular loop; ALIX depletion or mutation of the YPX3L motif prevents P2Y1 sorting into MVB lumen and degradation.","method":"RNAi knockdown, site-directed mutagenesis, MVB sorting assays","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 — knockdown plus mutagenesis of binding motif with specific MVB sorting readout, single lab","pmids":["27301021"],"is_preprint":false},{"year":2016,"finding":"Alix is required for clathrin-independent endocytosis (CIE) of fluid phase and specific cargo (cholera toxin) but not for clathrin-mediated endocytosis; Alix acts with endophilin-A to promote CIE, and is required for fast endocytosis and downstream signaling of the IL-2 receptor.","method":"Alix knockout cells, endocytosis assays, siRNA knockdown, signaling assays","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 — KO cells with specific cargo and signaling readouts, single lab","pmids":["27244115"],"is_preprint":false},{"year":2016,"finding":"Alix interacts with F-actin, the Par complex, and ZO-1 to mediate assembly of the apically restricted actomyosin–tight junction complex; Alix knockout in mice causes structural epithelial defects (asymmetric cell shape, misplaced cilia, blebbing microvilli), lateral ventricle enlargement, and hydrocephalus.","method":"Alix knockout mouse model, co-immunoprecipitation, immunofluorescence, electron microscopy","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — KO mouse with specific morphological phenotype plus Co-IP identifying the polarity complex","pmids":["27336173"],"is_preprint":false},{"year":2016,"finding":"Alix interacts with Ago2 (Argonaute 2) in extracellular vesicles; Alix knockdown decreases miRNA levels in EVs without affecting EV number, indicating Alix plays a role in miRNA enrichment/packaging during EV biogenesis.","method":"Co-immunoprecipitation, Alix siRNA knockdown, RT-qPCR of EV miRNAs","journal":"International journal of molecular medicine","confidence":"Medium","confidence_rationale":"Tier 3 — single Co-IP and loss-of-function with miRNA readout, single lab","pmids":["26935291"],"is_preprint":false},{"year":2018,"finding":"ALIX depletion causes defective PD-L1 trafficking through the MVB and reduces exosomal PD-L1 secretion, leading to redistribution of PD-L1 to the cell surface; ALIX also controls EGFR activity; these effects create an immunosuppressive phenotype dependent on EGFR signaling.","method":"siRNA screen, RNAi knockdown, PD-L1 trafficking assays, EGFR phosphorylation assays","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 — RNAi knockdown with specific trafficking and signaling readouts, single lab","pmids":["30021161"],"is_preprint":false},{"year":2018,"finding":"Alix undergoes S-palmitoylation (S-acylation), a lipid post-translational modification that influences its interaction with tetraspanin CD9 and maintains the structural organization of exosome-like small extracellular vesicle membranes.","method":"Biochemical S-palmitoylation assays, co-immunoprecipitation, biophysical vesicle membrane analysis","journal":"Biochimica et biophysica acta. General subjects","confidence":"Medium","confidence_rationale":"Tier 3 — PTM identified with functional interaction consequences, single lab","pmids":["30251702"],"is_preprint":false},{"year":2019,"finding":"During necroptosis, phosphorylated MLKL is removed from membranes by ALIX-syntenin-1-mediated exocytosis (as well as flotillin-mediated endocytosis); ALIX and syntenin-1 are found in MLKL membrane immunoprecipitates by protein cross-linking/affinity purification.","method":"Protein cross-linking, affinity purification, functional necroptosis assays","journal":"Science signaling","confidence":"Medium","confidence_rationale":"Tier 2 — cross-linking/AP identifies complex, functional role in membrane clearance established, single lab","pmids":["31138766"],"is_preprint":false},{"year":2019,"finding":"In Drosophila, ALIX is recruited to the cytokinetic midbody via its V domain interaction with centralspindlin component Pavarotti (MKLP1), through a LxxLF motif in Pavarotti binding the conserved hydrophobic pocket of the ALIX V domain (the same pocket used by viral YPXnL/LxxLF motifs); ALIX and ESCRT-III Shrub cooperate for abscission in germline stem cells.","method":"Co-immunoprecipitation, mutagenesis, Drosophila genetics, live imaging, cytokinesis assays","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 — mechanistic interaction mapping with domain mutagenesis and in vivo genetic validation","pmids":["31607533"],"is_preprint":false},{"year":2020,"finding":"An active/open form of ALIX efficiently recruits ESCRT-III proteins to endosomes independent of other ESCRTs but requires lysobisphosphatidic acid (LBPA) in vivo; this ALIX–ESCRT-III pathway can be reconstituted on supported lipid bilayers in vitro and promotes tetraspanin sorting to exosomes.","method":"In vitro reconstitution on supported bilayers, RNAi knockdown, endosome fractionation, immunofluorescence","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution plus RNAi loss-of-function with specific cargo sorting readout","pmids":["32049272"],"is_preprint":false},{"year":2020,"finding":"ALIX–syntenin–syndecan-4 constitutes a plasma membrane-to-ESCRT module at the cytokinetic abscission site; direct interactions among these three proteins are essential for proper recruitment of ESCRT-III machinery at the abscission site (not at the midbody); depletion of any of the three delays abscission.","method":"Proteomics (Flemmingsome), co-immunoprecipitation, RNAi knockdown, live imaging of abscission","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — proteomics discovery plus Co-IP and RNAi knockdown with specific abscission phenotype, multiple proteins validated","pmids":["32321914"],"is_preprint":false},{"year":2020,"finding":"ALIX PRD contains three tandem proline-rich motifs that compete for a single binding site on TSG101-UEV, as demonstrated by NMR relaxation dispersion; the C-terminal tyrosine-rich fragment of ALIX-PRD forms reversible amyloid fibrils that are dissolved by Src kinase-mediated hyperphosphorylation.","method":"Heteronuclear NMR spectroscopy, relaxation dispersion analysis, dye-binding assays (Congo Red, ThT), transmission electron microscopy, Src kinase in vitro phosphorylation","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with structural NMR and multiple orthogonal biophysical methods","pmids":["32917811"],"is_preprint":false},{"year":2020,"finding":"Alix and Syntenin-1 are essential for proper subcellular localization and efficient EV secretion of amyloid precursor protein (APP) via an ESCRT-independent pathway; knockdown of either protein alters APP localization to ER and endolysosomal compartments, respectively.","method":"Inducible gene knockdown, subcellular fractionation, EV isolation, immunofluorescence","journal":"BMC molecular and cell biology","confidence":"Medium","confidence_rationale":"Tier 2 — sequential knockdowns with specific subcellular localization and EV cargo readouts, single lab","pmids":["32731849"],"is_preprint":false},{"year":2023,"finding":"SUMOylated hnRNPA2B1 (at K108, driven by UBC9 upregulated by circTLCD4-RWDD3) is recognized by the SUMO-interaction motif (SIM) of ALIX, which activates ALIX to recruit ESCRT-III, facilitating sorting of circTLCD4-RWDD3 into extracellular vesicles; mutation of ALIX SIM or K108 of hnRNPA2B1 blocks EV packaging.","method":"Co-immunoprecipitation, mutagenesis of ALIX SIM, SUMO modification assays, EV cargo sorting assays, in vivo tumor models","journal":"Signal transduction and targeted therapy","confidence":"Medium","confidence_rationale":"Tier 2 — mechanistic Co-IP with SIM mutagenesis and functional EV sorting assay, single lab","pmids":["37925421"],"is_preprint":false}],"current_model":"ALIX (PDCD6IP) is an autoinhibited adaptor protein (held in a closed conformation by PRD-Bro1 intramolecular interaction) that, upon activation by Ca2+-bound ALG-2 or binding partners, exposes its Bro1 domain (which recruits CHMP4/ESCRT-III) and V domain (which binds YPX(n)L late-domain motifs of viruses, ubiquitinated receptors carrying YPX(n)L-like sequences, or K63-linked polyubiquitin chains) to scaffold a flexible, dimeric platform that links viral Gag proteins, activated receptors, syntenin-syndecan, and SUMO-modified cargo to the ESCRT-III membrane fission machinery, thereby driving topologically equivalent membrane budding events including HIV budding, MVB intraluminal vesicle formation/cargo sorting, exosome biogenesis, and cytokinetic abscission, while also participating in F-actin organization, epithelial polarity maintenance, ER stress signaling via IRE1, and regulation of EGFR/VEGFR trafficking and signaling."},"narrative":{"teleology":[{"year":1999,"claim":"The initial identification of ALIX as a Ca²⁺-dependent ALG-2-interacting protein established it as a cytosolic factor potentially linking calcium signaling to cell death pathways, raising the question of its molecular function.","evidence":"Yeast two-hybrid screen with ALG-2, colocalization, and overexpression protection from cell death","pmids":["9880530"],"confidence":"Medium","gaps":["No endogenous loss-of-function data","Mechanism of cell death protection unknown","Identity of physiological downstream targets not established"]},{"year":2003,"claim":"Discovery that ALIX links retroviral Gag late domains to ESCRT-III/Tsg101 and directly binds CHMP4b via its Bro1 domain established ALIX as a central adaptor in ESCRT-dependent membrane budding, redefining it from a death-related protein to a membrane trafficking factor.","evidence":"Co-immunoprecipitation, GST pulldown, virus budding assays across HIV-1 and EIAV; fluorescence colocalization with CHMP4b at endosomes","pmids":["14505569","12860994"],"confidence":"High","gaps":["Structural basis of ALIX–cargo and ALIX–CHMP4 interactions unknown","Endogenous cellular cargo not identified"]},{"year":2004,"claim":"Mapping of the ALG-2 binding site to ALIX's PRD PxY repeats, identification of ALIX as an inhibitor of EGFR internalization via antagonism of Cbl–CIN85, and the finding that Src phosphorylation regulates ALIX localization and interactions collectively revealed ALIX as a regulated, multi-domain scaffold at the interface of receptor trafficking and signaling.","evidence":"Mutagenesis mapping ALG-2 site (residues 794–827); siRNA knockdown affecting EGFR internalization; in vitro Src kinase assays with subcellular fractionation","pmids":["14999017","15456872","15557335"],"confidence":"Medium","gaps":["Whether Src phosphorylation regulates ALIX in physiological signaling contexts not shown","Relationship between ALG-2 binding and EGFR trafficking not clarified"]},{"year":2007,"claim":"Crystal structures of full-length ALIX domains revealed the Bro1–V architecture and defined the molecular basis of dual cargo recognition (YPX(n)L motifs on V domain, CHMP4 helices on Bro1 concave face), while functional studies established ALIX as essential for cytokinetic abscission through CEP55-mediated midbody recruitment.","evidence":"X-ray crystallography of Bro1 and V domains with viral peptides; mutagenesis validated in budding assays; CEP55 interaction by proteomics and Co-IP; RNAi abscission failure","pmids":["17350572","17277784","18066081","17853893","18641129"],"confidence":"High","gaps":["How ALIX activation is regulated at the midbody versus endosomes not distinguished","Full-length ALIX structure in autoinhibited state not solved"]},{"year":2008,"claim":"Atomic-resolution structures of Bro1–CHMP4 and ALG-2–ALIX peptide complexes defined the structural logic of both ESCRT-III recruitment and Ca²⁺-dependent activation, revealing the arginine-switch mechanism in ALG-2 that opens a hydrophobic pocket for ALIX binding.","evidence":"X-ray crystallography of Bro1–CHMP4A/B/C peptide complexes and ALG-2–ALIX(799–814) complex with mutagenesis validation","pmids":["18511562","18940611"],"confidence":"High","gaps":["Whether ALG-2 binding alone is sufficient to relieve autoinhibition not tested","Structure of full-length ALG-2–ALIX complex not determined"]},{"year":2009,"claim":"SAXS and mutagenesis demonstrated that ALIX functions as a crescent-shaped dimer that bridges CHMP4B filaments, establishing dimerization as a prerequisite for its membrane-remodeling functions.","evidence":"Small-angle X-ray scattering, dimerization-disrupting mutations impairing HIV-1 budding, in vitro CHMP4B filament bridging","pmids":["19523902"],"confidence":"High","gaps":["Whether dimerization is constitutive or regulated not resolved","Structural model of dimeric ALIX on membranes not available"]},{"year":2010,"claim":"Two independent studies demonstrated that ALIX is autoinhibited by intramolecular PRD–Bro1 and PRD–V domain contacts that mask both the CHMP4- and cargo-binding surfaces, resolving the question of how ALIX activity is kept in check until needed.","evidence":"Systematic truncation/mutagenesis showing PRD folds back against V domain and Bro1; destabilizing mutations increase membrane association and virus budding","pmids":["21715492","20929444"],"confidence":"High","gaps":["Kinetics and trigger of autoinhibition relief in cells not established","Whether different activators use different mechanisms to open ALIX not tested"]},{"year":2012,"claim":"ALIX was shown to mediate ubiquitin-independent MVB sorting of GPCRs (PAR1) via YPX(3)L motif recognition and to selectively bind K63-linked polyubiquitin chains, revealing parallel cargo-recognition mechanisms—motif-dependent and ubiquitin-dependent—that diversify ALIX's client repertoire.","evidence":"PAR1 YPX3L mutagenesis and ALIX RNAi blocking MVB sorting; in vitro K63-polyUb binding with V domain mutagenesis","pmids":["22547407","23201121"],"confidence":"High","gaps":["Whether K63-Ub and YPXnL binding are mutually exclusive on the V domain not clarified","Full scope of ALIX-dependent ubiquitin-independent cargos not catalogued"]},{"year":2014,"claim":"Identification of the syndecan–syntenin–ALIX axis as the principal pathway for exosome/ILV biogenesis extended ALIX function beyond viral budding and receptor sorting to a major physiological membrane budding pathway regulated by ARF6/PLD2 and heparanase.","evidence":"RNAi, Co-IP, nanoparticle tracking, and electron microscopy in multiple studies","pmids":["24637612","25732677"],"confidence":"High","gaps":["How LBPA cooperates with syntenin–ALIX at the membrane not mechanistically resolved","Relative contribution of syntenin–ALIX versus ESCRT-0/I/II to total ILV budding unknown"]},{"year":2015,"claim":"Ca²⁺-dependent ALG-2 binding was shown to fully relieve ALIX autoinhibition and promote CHMP4-dependent membrane association specifically for EGFR MVB sorting, while ALIX's role in cytokinesis and viral budding was ALG-2-independent, establishing context-specific activation mechanisms.","evidence":"Domain-specific mutagenesis with parallel readouts for EGFR sorting, cytokinesis, and EIAV budding","pmids":["27462417"],"confidence":"High","gaps":["What activates ALIX at the midbody if not ALG-2 remains unclear","ATG12–ATG3 as activator (PMID:25686249) not integrated with ALG-2 mechanism"]},{"year":2016,"claim":"Alix knockout mice revealed an unexpected role in epithelial polarity: ALIX interacts with F-actin, the Par polarity complex, and ZO-1 to organize apical actomyosin–tight junction complexes, and its loss causes hydrocephalus, demonstrating essential developmental functions beyond endosomal trafficking.","evidence":"Alix knockout mouse phenotype (hydrocephalus, epithelial defects), Co-IP with Par complex and ZO-1, electron microscopy","pmids":["27336173"],"confidence":"High","gaps":["Whether polarity defects are secondary to trafficking defects or reflect a direct structural role for ALIX not resolved","Which domain of ALIX mediates Par complex interaction not mapped"]},{"year":2020,"claim":"In vitro reconstitution on supported lipid bilayers showed that open/active ALIX independently recruits ESCRT-III to LBPA-containing membranes without requiring upstream ESCRTs, while the syndecan-4–syntenin–ALIX module was identified as the abscission-site ESCRT-III recruitment platform distinct from the midbody CEP55 pathway.","evidence":"Reconstitution on supported bilayers; Flemmingsome proteomics with Co-IP and live imaging of abscission","pmids":["32049272","32321914"],"confidence":"High","gaps":["Whether LBPA directly binds ALIX or acts indirectly not determined","Structural basis of syndecan-4–syntenin–ALIX ternary complex not solved"]},{"year":2020,"claim":"NMR studies revealed that the ALIX PRD contains three tandem motifs competing for a single TSG101-UEV site and that the tyrosine-rich PRD tail forms reversible amyloid fibrils dissolved by Src phosphorylation, suggesting a regulated phase-transition mechanism for ALIX PRD function.","evidence":"Heteronuclear NMR relaxation dispersion, Congo Red/ThT amyloid assays, TEM, in vitro Src phosphorylation","pmids":["32917811"],"confidence":"High","gaps":["Whether PRD amyloid formation occurs in cells not demonstrated","Functional consequence of amyloid dissolution on trafficking or budding not tested"]},{"year":2023,"claim":"Discovery that ALIX harbors a SUMO-interaction motif (SIM) that recognizes SUMOylated hnRNPA2B1 to sort circular RNA cargo into extracellular vesicles expanded the repertoire of ALIX cargo-recognition mechanisms beyond YPXnL motifs and ubiquitin.","evidence":"Co-IP, ALIX SIM mutagenesis, SUMO modification assays, EV cargo sorting in tumor models","pmids":["37925421"],"confidence":"Medium","gaps":["SIM-dependent sorting not validated with purified components","Generality of SUMO-dependent ALIX cargo recognition beyond this circRNA not explored","Single study, awaits independent confirmation"]},{"year":null,"claim":"A full-length structure of autoinhibited ALIX, the precise mechanism by which distinct activators (ALG-2, ATG12–ATG3, binding partners) differentially open ALIX for context-specific functions, and the in vivo relevance of PRD amyloid formation remain unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No full-length ALIX structure in closed or open state","Relative contribution of ALIX to total cellular ILV/exosome budding not quantified","How epithelial polarity and actin-organizing roles relate mechanistically to ESCRT-scaffolding function not integrated"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,2,10,13,26,30,45]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[9,28,38]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[20,38]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,7]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[2,8,44]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[30,44]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[9,28,38]},{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[30,39,41]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[1,2,26,30,44,45]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[13,15,43,45]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[4,32,33,40]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[1,11,14,24]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[6,42]}],"complexes":["ESCRT-III (via CHMP4B)","syndecan–syntenin–ALIX","CEP55–ALIX–TSG101 (midbody)"],"partners":["CHMP4B","TSG101","ALG2","CEP55","SDCBP","CIN85","ACTN1","CTTN"],"other_free_text":[]},"mechanistic_narrative":"ALIX (PDCD6IP) is a multifunctional ESCRT-associated adaptor protein that links cargo recognition to ESCRT-III-dependent membrane fission across topologically equivalent processes including multivesicular body (MVB) biogenesis, exosome secretion, retroviral budding, and cytokinetic abscission. ALIX exists in an autoinhibited closed conformation maintained by intramolecular contacts between its proline-rich domain (PRD) and Bro1 domain; Ca²⁺-bound ALG-2 or binding partners relieve this autoinhibition, exposing the Bro1 domain for CHMP4/ESCRT-III recruitment and the V domain for recognition of YPX(n)L motifs on viral Gag proteins and GPCRs, K63-linked polyubiquitin chains, and syntenin–syndecan complexes [PMID:22547407, PMID:23201121, PMID:17350572, PMID:20929444, PMID:27462417, PMID:24637612]. Active ALIX dimerizes via its V domain to bridge CHMP4B filaments, and this dimeric scaffold is required for HIV-1 budding, EGFR MVB sorting, and cytokinetic abscission where ALIX is recruited to the midbody by CEP55 and to the abscission site by syndecan-4–syntenin [PMID:19523902, PMID:17853893, PMID:32321914]. Beyond endosomal trafficking, ALIX directly binds F-actin, cortactin, and polarity determinants (Par complex, ZO-1) to organize apical actomyosin–tight junction complexes, and Alix knockout in mice causes epithelial polarity defects and hydrocephalus [PMID:27336173, PMID:16966331]."},"prefetch_data":{"uniprot":{"accession":"Q8WUM4","full_name":"Programmed cell death 6-interacting protein","aliases":["ALG-2-interacting protein 1","ALG-2-interacting protein X","Hp95"],"length_aa":868,"mass_kda":96.0,"function":"Multifunctional protein involved in endocytosis, multivesicular body biogenesis, membrane repair, cytokinesis, apoptosis and maintenance of tight junction integrity. Class E VPS protein involved in concentration and sorting of cargo proteins of the multivesicular body (MVB) for incorporation into intralumenal vesicles (ILVs) that are generated by invagination and scission from the limiting membrane of the endosome. Binds to the phospholipid lysobisphosphatidic acid (LBPA) which is abundant in MVBs internal membranes. The MVB pathway requires the sequential function of ESCRT-O, -I,-II and -III complexes (PubMed:14739459). The ESCRT machinery also functions in topologically equivalent membrane fission events, such as the terminal stages of cytokinesis (PubMed:17556548, PubMed:17853893). Adapter for a subset of ESCRT-III proteins, such as CHMP4, to function at distinct membranes. Required for completion of cytokinesis (PubMed:17556548, PubMed:17853893, PubMed:18641129). May play a role in the regulation of both apoptosis and cell proliferation. Regulates exosome biogenesis in concert with SDC1/4 and SDCBP (PubMed:22660413). By interacting with F-actin, PARD3 and TJP1 secures the proper assembly and positioning of actomyosin-tight junction complex at the apical sides of adjacent epithelial cells that defines a spatial membrane domain essential for the maintenance of epithelial cell polarity and barrier (By similarity) (Microbial infection) Involved in HIV-1 virus budding. Can replace TSG101 it its role of supporting HIV-1 release; this function requires the interaction with CHMP4B. The ESCRT machinery also functions in topologically equivalent membrane fission events, such as enveloped virus budding (HIV-1 and other lentiviruses)","subcellular_location":"Cytoplasm, cytosol; Melanosome; Cytoplasm, cytoskeleton, microtubule organizing center, centrosome; Secreted, extracellular exosome; Cell junction, tight junction; Midbody, Midbody ring","url":"https://www.uniprot.org/uniprotkb/Q8WUM4/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PDCD6IP","classification":"Not Classified","n_dependent_lines":527,"n_total_lines":1208,"dependency_fraction":0.43625827814569534},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000170248","cell_line_id":"CID000069","localizations":[{"compartment":"cytoplasmic","grade":3},{"compartment":"nucleoplasm","grade":1}],"interactors":[{"gene":"SCAMP2","stoichiometry":10.0},{"gene":"RPS6KA3","stoichiometry":4.0},{"gene":"PDCD6","stoichiometry":0.2},{"gene":"RNF2","stoichiometry":0.2},{"gene":"SYN2","stoichiometry":0.2},{"gene":"GEMIN5","stoichiometry":0.2},{"gene":"ARL8B","stoichiometry":0.2},{"gene":"CEP55","stoichiometry":0.2},{"gene":"SAR1B","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000069","total_profiled":1310},"omim":[{"mim_id":"620047","title":"MICROCEPHALY 29, PRIMARY, AUTOSOMAL RECESSIVE; MCPH29","url":"https://www.omim.org/entry/620047"},{"mim_id":"610897","title":"CHARGED MULTIVESICULAR BODY PROTEIN 4B; CHMP4B","url":"https://www.omim.org/entry/610897"},{"mim_id":"610051","title":"CHARGED MULTIVESICULAR BODY PROTEIN 4A; CHMP4A","url":"https://www.omim.org/entry/610051"},{"mim_id":"610000","title":"CENTROSOMAL PROTEIN, 55-KD; CEP55","url":"https://www.omim.org/entry/610000"},{"mim_id":"609787","title":"UBIQUITIN-ASSOCIATED PROTEIN 1; UBAP1","url":"https://www.omim.org/entry/609787"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Vesicles","reliability":"Approved"},{"location":"Centrosome","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PDCD6IP"},"hgnc":{"alias_symbol":["Alix","AIP1","Hp95"],"prev_symbol":[]},"alphafold":{"accession":"Q8WUM4","domains":[{"cath_id":"1.25.40.280","chopping":"12-329","consensus_level":"medium","plddt":93.3318,"start":12,"end":329},{"cath_id":"1.20.120.560","chopping":"364-400_542-640","consensus_level":"medium","plddt":91.9781,"start":364,"end":640},{"cath_id":"1.20.140.50","chopping":"428-539_649-713","consensus_level":"medium","plddt":92.8203,"start":428,"end":713}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8WUM4","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8WUM4-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8WUM4-F1-predicted_aligned_error_v6.png","plddt_mean":83.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PDCD6IP","jax_strain_url":"https://www.jax.org/strain/search?query=PDCD6IP"},"sequence":{"accession":"Q8WUM4","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8WUM4.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8WUM4/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8WUM4"}},"corpus_meta":[{"pmid":"14505569","id":"PMC_14505569","title":"AIP1/ALIX 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Overexpression of a truncated AIP1 protects cells from trophic factor withdrawal-induced death.\",\n      \"method\": \"Yeast two-hybrid screening, colocalization, overexpression assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — original identification with yeast two-hybrid and functional overexpression data, single lab\",\n      \"pmids\": [\"9880530\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"ALIX/AIP1 functions in HIV-1 and EIAV retrovirus budding by binding to a secondary late-domain (L-domain) region in HIV-1 Gag p6 and to the EIAV p9 L-domain; ALIX also interacts with Tsg101 and ESCRT-III homologs, serving to link viral Gag to the host ESCRT-III budding machinery.\",\n      \"method\": \"Co-immunoprecipitation, binding assays, virus budding assays\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, functional budding assays, replicated across retroviruses, highly cited\",\n      \"pmids\": [\"14505569\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"ALIX interacts directly with CHMP4b (a yeast Snf7 homolog and ESCRT-III component) via its N-terminal Bro1 domain, and this interaction was confirmed by GST pulldown and co-immunoprecipitation; both proteins colocalize at perinuclear endosomal structures and cooperate in multivesicular body (MVB) formation.\",\n      \"method\": \"Yeast two-hybrid, GST pulldown, co-immunoprecipitation, fluorescence microscopy\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (pulldown + Co-IP + localization), independently confirmed\",\n      \"pmids\": [\"12860994\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"The ALG-2-binding site on ALIX maps to amino acid residues 794–827 in the C-terminal proline-rich region, encompassing four PxY repeats; Ca2+-bound ALG-2 interaction with ALIX is required for the punctate subcellular distribution of the ALIX C-terminal fragment.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, alanine-scanning mutagenesis, fluorescence microscopy\",\n      \"journal\": \"Journal of biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis mapped binding site, orthogonal Co-IP and localization assays\",\n      \"pmids\": [\"14999017\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"ALIX/AIP1 inhibits EGFR internalization and downregulation by antagonizing the Cbl–CIN85/SETA complex; ALIX reduces Cbl-mediated ubiquitination and the interaction between Cbl and CIN85, while siRNA-mediated knockdown of Alix promotes EGFR internalization and degradation.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, receptor internalization and ubiquitination assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus loss-of-function with specific phenotypic readout, single lab\",\n      \"pmids\": [\"15456872\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Alix binds to the transferrin receptor (TfR) cytosolic YTRF motif and contributes to sorting of TfR into exosomes during reticulocyte maturation; AP2 degradation via the proteasome relieves competition at this site and allows hsc70 and Alix to drive TfR exosomal sorting.\",\n      \"method\": \"Co-immunoprecipitation, peptide affinity chromatography, proteasome inhibitor experiments\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP and pulldown, single lab with functional inhibitor data\",\n      \"pmids\": [\"15086793\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Paraptosis (a nonapoptotic cell death program induced by the IGF-I receptor) is specifically inhibited by AIP1/Alix, and this inhibition is distinct from apoptosis inhibition; the inhibition does not involve caspase pathways and is mediated via MAP kinases.\",\n      \"method\": \"Overexpression and dominant-negative AIP1, cell death assays, caspase activity assays\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean overexpression/dominant-negative with specific phenotypic readout, single lab\",\n      \"pmids\": [\"15195070\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Src kinase phosphorylates ALIX at its C-terminal tyrosine-rich region (via an SH3-SH2 interaction with ALIX), which causes ALIX to translocate from membrane/cytoskeleton to the cytoplasm and disrupts ALIX interactions with CIN85/SETA, EGFR, and Pyk2, antagonizing ALIX's inhibitory role in receptor internalization.\",\n      \"method\": \"In vitro kinase assays, co-immunoprecipitation, subcellular fractionation, overexpression/mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro kinase assay plus functional co-IP and localization data, single lab\",\n      \"pmids\": [\"15557335\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Depletion of Alix by RNAi causes redistribution of early endosomes from peripheral to perinuclear location and accumulation of unusual cortactin-containing actin structures, indicating Alix functions in actin-dependent intracellular positioning of endosomes; lysobisphosphatidic acid (LBPA) levels in MVEs are reduced upon Alix depletion.\",\n      \"method\": \"RNAi knockdown, immunofluorescence, immunoelectron microscopy\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — RNAi knockdown with specific morphological phenotype and quantitative EM, single lab\",\n      \"pmids\": [\"15914539\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"ALIX directly binds filamentous actin (F-actin) through both its N-terminal Bro1 domain and C-terminal proline-rich domain; in cells, ALIX also binds cortactin (via the N-terminal half of the V domain) and α-actinin (via the C-terminal half of the V domain), and is required for lamellipodial cortactin localization and stress fiber assembly.\",\n      \"method\": \"Cell-free actin-binding assays, co-immunoprecipitation, siRNA knockdown, immunofluorescence\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vitro binding plus Co-IP and RNAi functional assays, single lab\",\n      \"pmids\": [\"16966331\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Crystal structures of human ALIX reveal it is composed of an N-terminal Bro1 domain and a central V domain (two three-helix bundles forming elongated arms). YPX(n)L late-domain motifs of retroviruses bind a conserved hydrophobic pocket on the second arm of the V domain; CHMP4/ESCRT-III proteins bind a conserved hydrophobic patch on the Bro1 domain; both interactions are required for virus budding.\",\n      \"method\": \"X-ray crystallography, mutagenesis, virus budding assays\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structures with mutagenesis validation and functional budding assay\",\n      \"pmids\": [\"17350572\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The structure of the ALIX V domain bound to HIV-1 p6 LYPXnL motif was solved; Phe676 in a large hydrophobic pocket is crucial for binding; overexpression of the V domain alone inhibits HIV-1 release, and this is reversed by mutations blocking V domain–p6 interaction.\",\n      \"method\": \"X-ray crystallography, mutagenesis, virus budding inhibition assay\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with site-specific mutagenesis and functional rescue\",\n      \"pmids\": [\"17277784\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Crystal structures of ALIX V domain in complex with YPX(n)L late domains from HIV-1 and EIAV show that distinct late domains bind the same hydrophobic site on the V domain adopting different conformations; binding affinities are context-dependent and key interface residues were validated functionally.\",\n      \"method\": \"X-ray crystallography, surface plasmon resonance, mutagenesis, virus budding assays\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — dual crystal structures with biophysical binding measurements and functional assays\",\n      \"pmids\": [\"18066081\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"ALIX is recruited to the midbody of dividing cells through direct binding between the central CEP55 'hinge' region and a GPP-based motif in ALIX; depletion of ALIX (and TSG101) inhibits the abscission step of cytokinesis; ALIX point mutants blocking CEP55 and CHMP4/ESCRT-III binding also block abscission.\",\n      \"method\": \"Proteomics, co-immunoprecipitation, RNAi knockdown, dominant-negative mutants, live cell imaging\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods, replicated across studies, highly cited\",\n      \"pmids\": [\"17853893\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"ALIX potently rescues HIV-1 L-domain mutant budding; this rescue depends on intact ALIX–p6 binding site and the ESCRT-III (CHMP4B)-binding surface on the Bro1 domain; ALIX C-terminal proline-rich domain (PRD) is also required but independently of TSG101/endophilin/CIN85 binding sites.\",\n      \"method\": \"Mutagenesis, virus budding assays, co-immunoprecipitation\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — systematic mutagenesis with functional budding readouts\",\n      \"pmids\": [\"17428861\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"ALIX is recruited to the cytokinetic midbody via Cep55 interaction through an evolutionarily conserved peptide; disruption of Cep55/Alix/ESCRT-III interactions causes aberrant midbodies and cytokinetic failure; ALIX C-terminus encodes a multimerization activity essential for Alix-dependent HIV-1 release and Tsg101 interaction.\",\n      \"method\": \"Co-immunoprecipitation, dominant-negative mutants, siRNA knockdown, cytokinesis failure assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal assays establishing mechanistic links in both cytokinesis and virus budding\",\n      \"pmids\": [\"18641129\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"HIV-1 ALIX also engages the nucleocapsid (NC) domain of Gag via its Bro1 domain (zinc finger-dependent, RNA-independent) allowing ALIX incorporation into virions independently of the p6 L-domain; NC zinc finger mutations reduce particle production and show functional relationship with the p6 PTAP motif.\",\n      \"method\": \"Co-immunoprecipitation, nuclease treatment, HIV budding assays, ALIX overexpression rescue\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP with controls plus functional budding assays, single lab\",\n      \"pmids\": [\"18032513\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Crystal structures of the ALIX Bro1 domain bound to C-terminal peptides of CHMP4A-C reveal that CHMP4 C-terminal amphipathic helices bind across the concave surface of Bro1; key hydrophobic residues (M/L/IxxLxxW pattern) mediate binding; mutations in exposed Bro1 surface residues block ALIX-dependent HIV-1 budding.\",\n      \"method\": \"X-ray crystallography, mutagenesis, HIV-1 budding assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structures with mutagenesis and functional validation\",\n      \"pmids\": [\"18511562\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"ALG-2 crystal structure in complex with the Alix 799-814 peptide reveals a Ca2+/EF3-driven arginine switch: Ca2+ binding to EF3 moves Arg125 to open a hydrophobic pocket that accommodates the PPYP motif of Alix, explaining the Ca2+-dependence of ALG-2/Alix association.\",\n      \"method\": \"X-ray crystallography, in vitro binding assays with mutant proteins\",\n      \"journal\": \"Structure (London, England : 1993)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structures with mutagenesis validation of binding mechanism\",\n      \"pmids\": [\"18940611\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"AIP1 (DAB2IP) interacts with IRE1 via its PH domain and facilitates IRE1 dimerization, a critical step for IRE1 signaling; AIP1-deficient cells and mice show selectively impaired ER stress-induced IRE1-JNK/XBP-1 signaling but normal PERK-CHOP signaling.\",\n      \"method\": \"AIP1 knockout mice, co-immunoprecipitation, reconstitution with deletion mutants, in vivo signaling assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO mice plus domain-mapping co-IP and reconstitution, specific axis validated\",\n      \"pmids\": [\"18281285\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"ALIX dimerizes via its V domain in solution; SAXS modeling reveals a crescent-shaped dimeric conformation; dimeric ALIX colocalizes with and bridges CHMP4B filaments; dimerization-disrupting mutations impair HIV-1 budding, indicating dimeric ALIX is the active form that interacts with ESCRT-III CHMP4 polymers.\",\n      \"method\": \"SAXS, mutagenesis, co-localization, HIV-1 budding assay, in vitro CHMP4B filament bridging\",\n      \"journal\": \"Structure (London, England : 1993)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — structural analysis (SAXS) with mutagenesis and functional budding assays\",\n      \"pmids\": [\"19523902\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"ALG-2 acts as a Ca2+-dependent adaptor that bridges Alix and TSG101; the ALG-2-binding site on Alix (not the PSAP/TSG101-UEV binding site) is required for Ca2+-dependent TSG101 pulldown; ALG-2 dimerization and Ca2+ binding are both required for bridging.\",\n      \"method\": \"Strep-pulldown with mutants, ALG-2 knockdown, recombinant protein reconstitution\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — domain-mapping pulldown with KD and reconstitution, single lab\",\n      \"pmids\": [\"19520058\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The ALIX C-terminal proline-rich region (PRR) folds back against upstream domains and auto-inhibits V domain binding to viral late domains; mutations that destabilize this closed conformation open the V domain, increase ALIX membrane association, and enhance virus budding.\",\n      \"method\": \"Mutagenesis, in vitro binding assays, SAXS/structural modeling, virus budding assays\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — structural evidence of autoinhibition plus mutagenesis and functional budding assay\",\n      \"pmids\": [\"21715492\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"An intramolecular interaction between Patch 2 in the Bro1 domain and the TSG101-docking site in the ALIX PRD locks ALIX in a closed, autoinhibited conformation that prevents CHMP4 and retroviral Gag binding; relieving this intramolecular interaction (by expressing binding partners or deleting interaction sites) enables ALIX to interact with CHMP4 and facilitates membrane association and EIAV budding.\",\n      \"method\": \"ALIX truncation and mutagenesis, co-immunoprecipitation, EIAV budding assay\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — systematic domain mapping with functional virus budding readout, consistent with parallel structural data\",\n      \"pmids\": [\"20929444\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"SIVmac239 and SIVagmTan-1 possess divergent ALIX-binding late domains distinct from canonical YPX(n)L; crystal structures reveal that anchoring tyrosines and adjacent hydrophobic residues contact the same ALIX V domain hydrophobic pocket used by HIV-1/EIAV late domains.\",\n      \"method\": \"X-ray crystallography, late-domain mapping, virus budding assays\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structures defining binding mechanism\",\n      \"pmids\": [\"20962096\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Alix Bro1 domain Surface 1 (concave face, CHMP4 binding site), Surface 2 (narrow end), and a unique extended loop exposing Phe105 all independently contribute to ALIX's ability to stimulate HIV-1 budding, as established by mutagenesis guided by the BROX crystal structure.\",\n      \"method\": \"X-ray crystallography (BROX), ALIX mutagenesis, HIV-1 budding assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 — structure-guided mutagenesis with functional budding assay, single lab\",\n      \"pmids\": [\"22162750\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ALIX binds the V domain to a YPX(3)L motif in the GPCR PAR1 and mediates ubiquitin-independent ESCRT-III-dependent MVB/lysosomal sorting of PAR1, providing an alternative MVB sorting pathway that bypasses ubiquitin-binding ESCRTs.\",\n      \"method\": \"Co-immunoprecipitation, RNAi knockdown, mutagenesis of YPX3L motif, MVB sorting assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, site-directed mutagenesis and KD with specific MVB sorting readout\",\n      \"pmids\": [\"22547407\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ALIX is a Lys63-specific polyubiquitin binding protein; the V domain directly and selectively binds K63-linked polyUb chains (preference for chains ≥3 Ub) via two putative Ub-binding sites on a single α-helical surface; mutations in these sites impair polyUb binding and lentivirus budding.\",\n      \"method\": \"Affinity capture/mass spectrometry, in vitro binding assays, mutagenesis, virus budding assays\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro direct binding with mutagenesis plus functional virus budding assay\",\n      \"pmids\": [\"23201121\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Alix is ubiquitinated by the Ozz-E3 ligase in skeletal muscle; interaction with Ozz changes Alix conformation and promotes its ubiquitination, regulating Alix levels at cortactin-positive actin remodeling subcompartments; Alix knockdown reduces F-actin levels and impairs cell motility in myotubes.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, Ozz-/- mouse model, siRNA knockdown\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO model plus in-cell ubiquitination and functional actin/motility assays, single lab\",\n      \"pmids\": [\"22334701\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"AP-3 adaptor complex binds a tyrosine-based motif in the PAR1 cytoplasmic tail and functions upstream of ALIX to facilitate PAR1 lysosomal sorting; AP-3 promotes PAR1–ALIX interaction, demonstrating a sequential AP-3→ALIX→ESCRT-III pathway for ubiquitin-independent MVB sorting.\",\n      \"method\": \"Co-immunoprecipitation, RNAi knockdown, mutagenesis, receptor sorting assays\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — epistasis established by RNAi knockdown with specific receptor-sorting readout, single lab\",\n      \"pmids\": [\"22833563\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The syndecan–syntenin–ALIX pathway controls exosome biogenesis and intraluminal budding; ALIX interaction with syntenin and LBPA is required for ILV budding into MVBs; ARF6 and PLD2 regulate this pathway by controlling ILV budding.\",\n      \"method\": \"RNAi knockdown, co-immunoprecipitation, nanoparticle tracking, electron microscopy\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods, replicated across subsequent studies\",\n      \"pmids\": [\"24637612\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The ATG12–ATG3 conjugate interacts with Alix (PDCD6IP) and controls multiple Alix-dependent processes including late endosome distribution, exosome biogenesis, and viral budding; cells lacking ATG12–ATG3 phenocopy Alix loss-of-function in terms of basal autophagic flux and perinuclear late endosome accumulation.\",\n      \"method\": \"Co-immunoprecipitation, KO cell lines, functional assays for autophagy/exosomes/virus budding\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP and loss-of-function in multiple assays across distinct cellular processes, single lab\",\n      \"pmids\": [\"25686249\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Ca2+-dependent ALG-2 interaction with ALIX completely relieves ALIX's intramolecular autoinhibition and promotes CHMP4-dependent ALIX membrane association; EGFR activation increases ALG-2–ALIX interaction and ALIX membrane association; inhibiting ALG-2-mediated ALIX activation blocks MVB sorting of activated EGFR but not cytokinetic abscission or EIAV budding.\",\n      \"method\": \"Co-immunoprecipitation, mutagenesis, EGFR MVB sorting assays, domain-specific inhibition\",\n      \"journal\": \"Cell discovery\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic domain mapping with context-specific functional readouts, single lab with orthogonal assays\",\n      \"pmids\": [\"27462417\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"ALIX mediates MVB sorting and silencing (signal attenuation) of activated ubiquitinated EGFR via V domain interaction with ubiquitinated EGFR and Bro1 domain interaction with membrane-bound CHMP4; inhibiting either ALIX dimerization or CHMP4 interaction blocks EGFR MVB sorting and promotes sustained ERK1/2 activation.\",\n      \"method\": \"EGF stimulation, co-immunoprecipitation, siRNA knockdown, signaling assays\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — domain-specific inhibition with functional receptor sorting and signaling readout, single lab\",\n      \"pmids\": [\"25510652\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Heparanase stimulates the syndecan–syntenin–ALIX exosome pathway by trimming heparan sulfate chains on syndecans, fostering endosomal membrane budding; exosomal secretion of syntenin-1, syndecan, and CD63 depends on the syntenin–ALIX interaction.\",\n      \"method\": \"RNAi knockdown, exosome quantification, co-immunoprecipitation, electron microscopy\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic epistasis established by syntenin/ALIX KD in heparanase-dependent pathway, single lab\",\n      \"pmids\": [\"25732677\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Nedd4-1 ubiquitin ligase interacts with ALIX (via Co-IP and yeast two-hybrid) and is recruited to facilitate HIV-1 release through the LYPXnL/ALIX pathway; Nedd4-1 catalytic activity ubiquitinates ALIX itself, and RNAi knockdown of Nedd4-1 eliminates ALIX ubiquitination and impairs ALIX-dependent HIV-1 release.\",\n      \"method\": \"Co-immunoprecipitation, yeast two-hybrid, RNAi knockdown, ubiquitination assay, virus budding assays\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods identifying Nedd4-1 as ALIX writer and functional role in budding, single lab\",\n      \"pmids\": [\"20519395\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ALIX mediates ubiquitin-independent lysosomal sorting of the P2Y1 purinergic GPCR through a YPX3L motif in the receptor's second intracellular loop; ALIX depletion or mutation of the YPX3L motif prevents P2Y1 sorting into MVB lumen and degradation.\",\n      \"method\": \"RNAi knockdown, site-directed mutagenesis, MVB sorting assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — knockdown plus mutagenesis of binding motif with specific MVB sorting readout, single lab\",\n      \"pmids\": [\"27301021\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Alix is required for clathrin-independent endocytosis (CIE) of fluid phase and specific cargo (cholera toxin) but not for clathrin-mediated endocytosis; Alix acts with endophilin-A to promote CIE, and is required for fast endocytosis and downstream signaling of the IL-2 receptor.\",\n      \"method\": \"Alix knockout cells, endocytosis assays, siRNA knockdown, signaling assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO cells with specific cargo and signaling readouts, single lab\",\n      \"pmids\": [\"27244115\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Alix interacts with F-actin, the Par complex, and ZO-1 to mediate assembly of the apically restricted actomyosin–tight junction complex; Alix knockout in mice causes structural epithelial defects (asymmetric cell shape, misplaced cilia, blebbing microvilli), lateral ventricle enlargement, and hydrocephalus.\",\n      \"method\": \"Alix knockout mouse model, co-immunoprecipitation, immunofluorescence, electron microscopy\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO mouse with specific morphological phenotype plus Co-IP identifying the polarity complex\",\n      \"pmids\": [\"27336173\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Alix interacts with Ago2 (Argonaute 2) in extracellular vesicles; Alix knockdown decreases miRNA levels in EVs without affecting EV number, indicating Alix plays a role in miRNA enrichment/packaging during EV biogenesis.\",\n      \"method\": \"Co-immunoprecipitation, Alix siRNA knockdown, RT-qPCR of EV miRNAs\",\n      \"journal\": \"International journal of molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP and loss-of-function with miRNA readout, single lab\",\n      \"pmids\": [\"26935291\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"ALIX depletion causes defective PD-L1 trafficking through the MVB and reduces exosomal PD-L1 secretion, leading to redistribution of PD-L1 to the cell surface; ALIX also controls EGFR activity; these effects create an immunosuppressive phenotype dependent on EGFR signaling.\",\n      \"method\": \"siRNA screen, RNAi knockdown, PD-L1 trafficking assays, EGFR phosphorylation assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — RNAi knockdown with specific trafficking and signaling readouts, single lab\",\n      \"pmids\": [\"30021161\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Alix undergoes S-palmitoylation (S-acylation), a lipid post-translational modification that influences its interaction with tetraspanin CD9 and maintains the structural organization of exosome-like small extracellular vesicle membranes.\",\n      \"method\": \"Biochemical S-palmitoylation assays, co-immunoprecipitation, biophysical vesicle membrane analysis\",\n      \"journal\": \"Biochimica et biophysica acta. General subjects\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — PTM identified with functional interaction consequences, single lab\",\n      \"pmids\": [\"30251702\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"During necroptosis, phosphorylated MLKL is removed from membranes by ALIX-syntenin-1-mediated exocytosis (as well as flotillin-mediated endocytosis); ALIX and syntenin-1 are found in MLKL membrane immunoprecipitates by protein cross-linking/affinity purification.\",\n      \"method\": \"Protein cross-linking, affinity purification, functional necroptosis assays\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — cross-linking/AP identifies complex, functional role in membrane clearance established, single lab\",\n      \"pmids\": [\"31138766\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"In Drosophila, ALIX is recruited to the cytokinetic midbody via its V domain interaction with centralspindlin component Pavarotti (MKLP1), through a LxxLF motif in Pavarotti binding the conserved hydrophobic pocket of the ALIX V domain (the same pocket used by viral YPXnL/LxxLF motifs); ALIX and ESCRT-III Shrub cooperate for abscission in germline stem cells.\",\n      \"method\": \"Co-immunoprecipitation, mutagenesis, Drosophila genetics, live imaging, cytokinesis assays\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic interaction mapping with domain mutagenesis and in vivo genetic validation\",\n      \"pmids\": [\"31607533\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"An active/open form of ALIX efficiently recruits ESCRT-III proteins to endosomes independent of other ESCRTs but requires lysobisphosphatidic acid (LBPA) in vivo; this ALIX–ESCRT-III pathway can be reconstituted on supported lipid bilayers in vitro and promotes tetraspanin sorting to exosomes.\",\n      \"method\": \"In vitro reconstitution on supported bilayers, RNAi knockdown, endosome fractionation, immunofluorescence\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution plus RNAi loss-of-function with specific cargo sorting readout\",\n      \"pmids\": [\"32049272\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ALIX–syntenin–syndecan-4 constitutes a plasma membrane-to-ESCRT module at the cytokinetic abscission site; direct interactions among these three proteins are essential for proper recruitment of ESCRT-III machinery at the abscission site (not at the midbody); depletion of any of the three delays abscission.\",\n      \"method\": \"Proteomics (Flemmingsome), co-immunoprecipitation, RNAi knockdown, live imaging of abscission\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — proteomics discovery plus Co-IP and RNAi knockdown with specific abscission phenotype, multiple proteins validated\",\n      \"pmids\": [\"32321914\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ALIX PRD contains three tandem proline-rich motifs that compete for a single binding site on TSG101-UEV, as demonstrated by NMR relaxation dispersion; the C-terminal tyrosine-rich fragment of ALIX-PRD forms reversible amyloid fibrils that are dissolved by Src kinase-mediated hyperphosphorylation.\",\n      \"method\": \"Heteronuclear NMR spectroscopy, relaxation dispersion analysis, dye-binding assays (Congo Red, ThT), transmission electron microscopy, Src kinase in vitro phosphorylation\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with structural NMR and multiple orthogonal biophysical methods\",\n      \"pmids\": [\"32917811\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Alix and Syntenin-1 are essential for proper subcellular localization and efficient EV secretion of amyloid precursor protein (APP) via an ESCRT-independent pathway; knockdown of either protein alters APP localization to ER and endolysosomal compartments, respectively.\",\n      \"method\": \"Inducible gene knockdown, subcellular fractionation, EV isolation, immunofluorescence\",\n      \"journal\": \"BMC molecular and cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — sequential knockdowns with specific subcellular localization and EV cargo readouts, single lab\",\n      \"pmids\": [\"32731849\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"SUMOylated hnRNPA2B1 (at K108, driven by UBC9 upregulated by circTLCD4-RWDD3) is recognized by the SUMO-interaction motif (SIM) of ALIX, which activates ALIX to recruit ESCRT-III, facilitating sorting of circTLCD4-RWDD3 into extracellular vesicles; mutation of ALIX SIM or K108 of hnRNPA2B1 blocks EV packaging.\",\n      \"method\": \"Co-immunoprecipitation, mutagenesis of ALIX SIM, SUMO modification assays, EV cargo sorting assays, in vivo tumor models\",\n      \"journal\": \"Signal transduction and targeted therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic Co-IP with SIM mutagenesis and functional EV sorting assay, single lab\",\n      \"pmids\": [\"37925421\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ALIX (PDCD6IP) is an autoinhibited adaptor protein (held in a closed conformation by PRD-Bro1 intramolecular interaction) that, upon activation by Ca2+-bound ALG-2 or binding partners, exposes its Bro1 domain (which recruits CHMP4/ESCRT-III) and V domain (which binds YPX(n)L late-domain motifs of viruses, ubiquitinated receptors carrying YPX(n)L-like sequences, or K63-linked polyubiquitin chains) to scaffold a flexible, dimeric platform that links viral Gag proteins, activated receptors, syntenin-syndecan, and SUMO-modified cargo to the ESCRT-III membrane fission machinery, thereby driving topologically equivalent membrane budding events including HIV budding, MVB intraluminal vesicle formation/cargo sorting, exosome biogenesis, and cytokinetic abscission, while also participating in F-actin organization, epithelial polarity maintenance, ER stress signaling via IRE1, and regulation of EGFR/VEGFR trafficking and signaling.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"ALIX (PDCD6IP) is a multifunctional ESCRT-associated adaptor protein that links cargo recognition to ESCRT-III-dependent membrane fission across topologically equivalent processes including multivesicular body (MVB) biogenesis, exosome secretion, retroviral budding, and cytokinetic abscission. ALIX exists in an autoinhibited closed conformation maintained by intramolecular contacts between its proline-rich domain (PRD) and Bro1 domain; Ca²⁺-bound ALG-2 or binding partners relieve this autoinhibition, exposing the Bro1 domain for CHMP4/ESCRT-III recruitment and the V domain for recognition of YPX(n)L motifs on viral Gag proteins and GPCRs, K63-linked polyubiquitin chains, and syntenin–syndecan complexes [PMID:22547407, PMID:23201121, PMID:17350572, PMID:20929444, PMID:27462417, PMID:24637612]. Active ALIX dimerizes via its V domain to bridge CHMP4B filaments, and this dimeric scaffold is required for HIV-1 budding, EGFR MVB sorting, and cytokinetic abscission where ALIX is recruited to the midbody by CEP55 and to the abscission site by syndecan-4–syntenin [PMID:19523902, PMID:17853893, PMID:32321914]. Beyond endosomal trafficking, ALIX directly binds F-actin, cortactin, and polarity determinants (Par complex, ZO-1) to organize apical actomyosin–tight junction complexes, and Alix knockout in mice causes epithelial polarity defects and hydrocephalus [PMID:27336173, PMID:16966331].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"The initial identification of ALIX as a Ca²⁺-dependent ALG-2-interacting protein established it as a cytosolic factor potentially linking calcium signaling to cell death pathways, raising the question of its molecular function.\",\n      \"evidence\": \"Yeast two-hybrid screen with ALG-2, colocalization, and overexpression protection from cell death\",\n      \"pmids\": [\"9880530\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No endogenous loss-of-function data\", \"Mechanism of cell death protection unknown\", \"Identity of physiological downstream targets not established\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Discovery that ALIX links retroviral Gag late domains to ESCRT-III/Tsg101 and directly binds CHMP4b via its Bro1 domain established ALIX as a central adaptor in ESCRT-dependent membrane budding, redefining it from a death-related protein to a membrane trafficking factor.\",\n      \"evidence\": \"Co-immunoprecipitation, GST pulldown, virus budding assays across HIV-1 and EIAV; fluorescence colocalization with CHMP4b at endosomes\",\n      \"pmids\": [\"14505569\", \"12860994\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of ALIX–cargo and ALIX–CHMP4 interactions unknown\", \"Endogenous cellular cargo not identified\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Mapping of the ALG-2 binding site to ALIX's PRD PxY repeats, identification of ALIX as an inhibitor of EGFR internalization via antagonism of Cbl–CIN85, and the finding that Src phosphorylation regulates ALIX localization and interactions collectively revealed ALIX as a regulated, multi-domain scaffold at the interface of receptor trafficking and signaling.\",\n      \"evidence\": \"Mutagenesis mapping ALG-2 site (residues 794–827); siRNA knockdown affecting EGFR internalization; in vitro Src kinase assays with subcellular fractionation\",\n      \"pmids\": [\"14999017\", \"15456872\", \"15557335\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether Src phosphorylation regulates ALIX in physiological signaling contexts not shown\", \"Relationship between ALG-2 binding and EGFR trafficking not clarified\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Crystal structures of full-length ALIX domains revealed the Bro1–V architecture and defined the molecular basis of dual cargo recognition (YPX(n)L motifs on V domain, CHMP4 helices on Bro1 concave face), while functional studies established ALIX as essential for cytokinetic abscission through CEP55-mediated midbody recruitment.\",\n      \"evidence\": \"X-ray crystallography of Bro1 and V domains with viral peptides; mutagenesis validated in budding assays; CEP55 interaction by proteomics and Co-IP; RNAi abscission failure\",\n      \"pmids\": [\"17350572\", \"17277784\", \"18066081\", \"17853893\", \"18641129\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How ALIX activation is regulated at the midbody versus endosomes not distinguished\", \"Full-length ALIX structure in autoinhibited state not solved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Atomic-resolution structures of Bro1–CHMP4 and ALG-2–ALIX peptide complexes defined the structural logic of both ESCRT-III recruitment and Ca²⁺-dependent activation, revealing the arginine-switch mechanism in ALG-2 that opens a hydrophobic pocket for ALIX binding.\",\n      \"evidence\": \"X-ray crystallography of Bro1–CHMP4A/B/C peptide complexes and ALG-2–ALIX(799–814) complex with mutagenesis validation\",\n      \"pmids\": [\"18511562\", \"18940611\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ALG-2 binding alone is sufficient to relieve autoinhibition not tested\", \"Structure of full-length ALG-2–ALIX complex not determined\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"SAXS and mutagenesis demonstrated that ALIX functions as a crescent-shaped dimer that bridges CHMP4B filaments, establishing dimerization as a prerequisite for its membrane-remodeling functions.\",\n      \"evidence\": \"Small-angle X-ray scattering, dimerization-disrupting mutations impairing HIV-1 budding, in vitro CHMP4B filament bridging\",\n      \"pmids\": [\"19523902\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether dimerization is constitutive or regulated not resolved\", \"Structural model of dimeric ALIX on membranes not available\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Two independent studies demonstrated that ALIX is autoinhibited by intramolecular PRD–Bro1 and PRD–V domain contacts that mask both the CHMP4- and cargo-binding surfaces, resolving the question of how ALIX activity is kept in check until needed.\",\n      \"evidence\": \"Systematic truncation/mutagenesis showing PRD folds back against V domain and Bro1; destabilizing mutations increase membrane association and virus budding\",\n      \"pmids\": [\"21715492\", \"20929444\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinetics and trigger of autoinhibition relief in cells not established\", \"Whether different activators use different mechanisms to open ALIX not tested\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"ALIX was shown to mediate ubiquitin-independent MVB sorting of GPCRs (PAR1) via YPX(3)L motif recognition and to selectively bind K63-linked polyubiquitin chains, revealing parallel cargo-recognition mechanisms—motif-dependent and ubiquitin-dependent—that diversify ALIX's client repertoire.\",\n      \"evidence\": \"PAR1 YPX3L mutagenesis and ALIX RNAi blocking MVB sorting; in vitro K63-polyUb binding with V domain mutagenesis\",\n      \"pmids\": [\"22547407\", \"23201121\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether K63-Ub and YPXnL binding are mutually exclusive on the V domain not clarified\", \"Full scope of ALIX-dependent ubiquitin-independent cargos not catalogued\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identification of the syndecan–syntenin–ALIX axis as the principal pathway for exosome/ILV biogenesis extended ALIX function beyond viral budding and receptor sorting to a major physiological membrane budding pathway regulated by ARF6/PLD2 and heparanase.\",\n      \"evidence\": \"RNAi, Co-IP, nanoparticle tracking, and electron microscopy in multiple studies\",\n      \"pmids\": [\"24637612\", \"25732677\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How LBPA cooperates with syntenin–ALIX at the membrane not mechanistically resolved\", \"Relative contribution of syntenin–ALIX versus ESCRT-0/I/II to total ILV budding unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Ca²⁺-dependent ALG-2 binding was shown to fully relieve ALIX autoinhibition and promote CHMP4-dependent membrane association specifically for EGFR MVB sorting, while ALIX's role in cytokinesis and viral budding was ALG-2-independent, establishing context-specific activation mechanisms.\",\n      \"evidence\": \"Domain-specific mutagenesis with parallel readouts for EGFR sorting, cytokinesis, and EIAV budding\",\n      \"pmids\": [\"27462417\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"What activates ALIX at the midbody if not ALG-2 remains unclear\", \"ATG12–ATG3 as activator (PMID:25686249) not integrated with ALG-2 mechanism\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Alix knockout mice revealed an unexpected role in epithelial polarity: ALIX interacts with F-actin, the Par polarity complex, and ZO-1 to organize apical actomyosin–tight junction complexes, and its loss causes hydrocephalus, demonstrating essential developmental functions beyond endosomal trafficking.\",\n      \"evidence\": \"Alix knockout mouse phenotype (hydrocephalus, epithelial defects), Co-IP with Par complex and ZO-1, electron microscopy\",\n      \"pmids\": [\"27336173\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether polarity defects are secondary to trafficking defects or reflect a direct structural role for ALIX not resolved\", \"Which domain of ALIX mediates Par complex interaction not mapped\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"In vitro reconstitution on supported lipid bilayers showed that open/active ALIX independently recruits ESCRT-III to LBPA-containing membranes without requiring upstream ESCRTs, while the syndecan-4–syntenin–ALIX module was identified as the abscission-site ESCRT-III recruitment platform distinct from the midbody CEP55 pathway.\",\n      \"evidence\": \"Reconstitution on supported bilayers; Flemmingsome proteomics with Co-IP and live imaging of abscission\",\n      \"pmids\": [\"32049272\", \"32321914\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether LBPA directly binds ALIX or acts indirectly not determined\", \"Structural basis of syndecan-4–syntenin–ALIX ternary complex not solved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"NMR studies revealed that the ALIX PRD contains three tandem motifs competing for a single TSG101-UEV site and that the tyrosine-rich PRD tail forms reversible amyloid fibrils dissolved by Src phosphorylation, suggesting a regulated phase-transition mechanism for ALIX PRD function.\",\n      \"evidence\": \"Heteronuclear NMR relaxation dispersion, Congo Red/ThT amyloid assays, TEM, in vitro Src phosphorylation\",\n      \"pmids\": [\"32917811\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PRD amyloid formation occurs in cells not demonstrated\", \"Functional consequence of amyloid dissolution on trafficking or budding not tested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Discovery that ALIX harbors a SUMO-interaction motif (SIM) that recognizes SUMOylated hnRNPA2B1 to sort circular RNA cargo into extracellular vesicles expanded the repertoire of ALIX cargo-recognition mechanisms beyond YPXnL motifs and ubiquitin.\",\n      \"evidence\": \"Co-IP, ALIX SIM mutagenesis, SUMO modification assays, EV cargo sorting in tumor models\",\n      \"pmids\": [\"37925421\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"SIM-dependent sorting not validated with purified components\", \"Generality of SUMO-dependent ALIX cargo recognition beyond this circRNA not explored\", \"Single study, awaits independent confirmation\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A full-length structure of autoinhibited ALIX, the precise mechanism by which distinct activators (ALG-2, ATG12–ATG3, binding partners) differentially open ALIX for context-specific functions, and the in vivo relevance of PRD amyloid formation remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No full-length ALIX structure in closed or open state\", \"Relative contribution of ALIX to total cellular ILV/exosome budding not quantified\", \"How epithelial polarity and actin-organizing roles relate mechanistically to ESCRT-scaffolding function not integrated\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 2, 10, 13, 26, 30, 45]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [9, 28, 38]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [20, 38]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 7]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [2, 8, 44]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [30, 44]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [9, 28, 38]},\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [30, 39, 41]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [1, 2, 26, 30, 44, 45]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [13, 15, 43, 45]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [4, 32, 33, 40]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [1, 11, 14, 24]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [6, 42]}\n    ],\n    \"complexes\": [\n      \"ESCRT-III (via CHMP4B)\",\n      \"syndecan–syntenin–ALIX\",\n      \"CEP55–ALIX–TSG101 (midbody)\"\n    ],\n    \"partners\": [\n      \"CHMP4B\",\n      \"TSG101\",\n      \"ALG2\",\n      \"CEP55\",\n      \"SDCBP\",\n      \"CIN85\",\n      \"ACTN1\",\n      \"CTTN\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}