{"gene":"PDCD6IP","run_date":"2026-06-10T05:19:53","timeline":{"discoveries":[{"year":1999,"finding":"Alix (PDCD6IP) was identified as a novel binding partner of ALG-2 (apoptosis-linked gene 2) in a calcium-dependent manner, established by yeast two-hybrid screening and co-immunoprecipitation experiments.","method":"Yeast two-hybrid screen; co-immunoprecipitation","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP confirming yeast two-hybrid, single lab, two orthogonal methods","pmids":["10200558"],"is_preprint":false},{"year":2002,"finding":"Alix binds endophilins (SH3p4, SH3p8, SH3p13) through a proline-rich domain interaction (PXRPPPP consensus) at its C-terminal half, demonstrated by yeast two-hybrid, co-immunoprecipitation, and overlay experiments; overexpression of Alix-CT causes cytoplasmic vacuolization enhanced by endophilin co-expression.","method":"Yeast two-hybrid; co-immunoprecipitation; overlay assay; cell transfection/overexpression","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP and overlay assays, single lab, multiple orthogonal methods","pmids":["12034747"],"is_preprint":false},{"year":2003,"finding":"HIV-1 Gag p6 contains a second L-domain region that binds AIP1/Alix (PDCD6IP); Alix also interacts with Tsg101 and ESCRT-III homologs, and binds the EIAV p9 L domain, identifying Alix as a component of the viral budding machinery linking retroviral Gag proteins to ESCRT-III.","method":"Co-immunoprecipitation; binding assays; functional budding assays","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP with multiple partners, functional validation of budding, independently replicated across labs","pmids":["14505569"],"is_preprint":false},{"year":2003,"finding":"Alix interacts with CHMP4b (human Snf7 homolog) as demonstrated by GST pull-down and co-immunoprecipitation; CHMP4b and Alix co-localize at perinuclear puncta overlapping with endosomal markers, and cooperate in multivesicular body formation.","method":"Yeast two-hybrid; GST pull-down; co-immunoprecipitation; fluorescence microscopy","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro pull-down plus co-IP plus localization, replicated by multiple labs","pmids":["12860994"],"is_preprint":false},{"year":2003,"finding":"Both fungal PalA and mammalian AIP1/Alix recognize the YPXL/I protein-protein binding motif, where Tyr, Pro, and Leu/Ile are crucial for binding, established by mutagenesis of the motif in PacC substrates and binding assays.","method":"Binding assays; site-directed mutagenesis of YPXL/I motif","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis plus binding assay, single lab","pmids":["12588984"],"is_preprint":false},{"year":2004,"finding":"Paraptosis (non-apoptotic cell death induced by IGFIR) is inhibited specifically by AIP-1/Alix overexpression; this inhibition does not affect apoptosis and is MAPK/JNK-1-mediated, placing Alix as a negative regulator of IGFIR-induced paraptosis.","method":"Cell transfection/overexpression; antisense oligonucleotides; MEK-2 inhibitors; cell death assays","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — functional knockdown/overexpression with specific phenotypic readout, single lab","pmids":["15195070"],"is_preprint":false},{"year":2004,"finding":"ALG-2 interacts with the C-terminal proline-rich region of Alix (residues 794–827) through four tandem PxY repeats in a Ca2+-dependent manner; specific proline and tyrosine residues within this region are critical for binding affinity, demonstrated by yeast two-hybrid, overlay assay, and co-immunoprecipitation with deletion/alanine-substitution mutants.","method":"Yeast two-hybrid; biotin-tagged overlay assay; co-immunoprecipitation; alanine mutagenesis","journal":"Journal of biochemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — mutagenesis combined with co-IP and overlay, multiple methods, defines binding region precisely","pmids":["14999017"],"is_preprint":false},{"year":2004,"finding":"Alix/AIP1 antagonizes EGFR downregulation by attenuating the Cbl-SETA/CIN85 interaction and inhibiting Cbl-mediated ubiquitination of EGFR; siRNA knockdown of Alix promotes EGFR internalization and downregulation.","method":"Co-immunoprecipitation; siRNA knockdown; ubiquitination assays; receptor internalization assay","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA plus co-IP plus ubiquitination assay, single lab, multiple methods","pmids":["15456872"],"is_preprint":false},{"year":2004,"finding":"Alix co-immunoprecipitates with exosomal transferrin receptor, and a peptide (P1, YTRFSLARQV) from the TfR cytosolic domain binds both hsc70 and Alix (via homology to its fungal homolog PalA), suggesting Alix contributes to exosomal sorting of TfR via the YTRF motif.","method":"Co-immunoprecipitation; peptide binding/affinity chromatography","journal":"Traffic (Copenhagen, Denmark)","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single co-IP and peptide binding, indirect evidence for YTRF interaction","pmids":["15086793"],"is_preprint":false},{"year":2004,"finding":"Src phosphorylates Alix at its C-terminal tyrosine-rich region; this phosphorylation causes Alix to translocate from membrane/cytoskeleton to cytoplasm and reduces its interactions with SETA/CIN85, EGFR, and Pyk2, antagonizing Alix's negative regulation of receptor internalization and cell adhesion.","method":"In vitro kinase assay; co-immunoprecipitation; subcellular fractionation; siRNA/overexpression","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro kinase assay plus co-IP plus localization, single lab","pmids":["15557335"],"is_preprint":false},{"year":2005,"finding":"Alix depletion by RNAi causes redistribution of early endosomes from peripheral to perinuclear location, accumulation of unusual actin structures containing clathrin and cortactin, and reduction of lysobisphosphatidic acid levels in multivesicular endosomes, without affecting transferrin recycling or EGF receptor degradation.","method":"RNAi knockdown; immunofluorescence microscopy; quantitative immunoelectron microscopy","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA with defined phenotypic readouts, multiple imaging methods, single lab","pmids":["15914539"],"is_preprint":false},{"year":2006,"finding":"Alix directly interacts with F-actin (via both Bro1 and proline-rich domains), cortactin (via the N-terminal half of the middle region), and alpha-actinin (via the C-terminal half of the middle region) in cell-free systems; Alix knockdown decreases F-actin content and inhibits stress fiber assembly and lamellipodial localization of cortactin.","method":"In vitro direct binding (cell-free); co-immunoprecipitation; siRNA knockdown; fluorescence microscopy","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — in vitro direct binding assay plus siRNA functional phenotype, single lab","pmids":["16966331"],"is_preprint":false},{"year":2007,"finding":"Crystal structures of ALIX reveal an N-terminal Bro1 domain and a central V domain composed of two three-helix bundle arms; YPX(n)L late domains 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; functional budding assays","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with mutagenesis and functional validation in a single rigorous study","pmids":["17350572"],"is_preprint":false},{"year":2007,"finding":"ALIX and TSG101/ESCRT-I are recruited to midbodies of dividing cells through direct interactions between CEP55 and GPP-based motifs within TSG101 and ALIX; ALIX point mutants blocking CEP55 and CHMP4/ESCRT-III binding inhibit cytokinetic abscission; depletion of ALIX inhibits the abscission step of HeLa cell cytokinesis.","method":"Proteomics; co-immunoprecipitation; siRNA depletion; fluorescence microscopy; cytokinesis assay","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, siRNA depletion with defined abscission phenotype, mutagenesis, replicated by other labs","pmids":["17853893"],"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 reveal that both bind the same hydrophobic pocket but adopt different conformations; binding affinity and budding function require key interface residues identified by mutagenesis.","method":"X-ray crystallography; surface plasmon resonance; mutagenesis; functional budding assays","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structures with mutagenesis and biophysical binding measurements in one rigorous study","pmids":["17277784","18066081"],"is_preprint":false},{"year":2007,"finding":"ALIX also engages HIV-1 Gag through the nucleocapsid (NC) domain via the Bro1 domain (in addition to the V domain/p6 interaction); this NC-Bro1 interaction depends on NC zinc fingers and allows ALIX packaging into viral particles; zinc finger mutations affect particle production similarly to PTAP deletion.","method":"Co-immunoprecipitation; viral packaging assays; nuclease treatment; mutagenesis","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP with mutagenesis and functional assay, single lab","pmids":["18032513"],"is_preprint":false},{"year":2008,"finding":"Crystal structures of ALIX Bro1 domain in complex with C-terminal peptides of CHMP4A-C show that CHMP4 C-terminal peptides form amphipathic helices binding across the concave surface of Bro1; a hydrophobic code (M/L/IxxLxxW) distinguishes CHMP4 binding to Bro1-domain proteins from CHMP1-3 binding to MIT-domain proteins (VPS4); mutations in CHMP4 recognition residues block ALIX-dependent HIV-1 budding.","method":"X-ray crystallography; mutagenesis; HIV-1 budding assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with mutagenesis and functional validation in one rigorous study","pmids":["18511562"],"is_preprint":false},{"year":2008,"finding":"ALG-2 binds Ca2+ through EF3, triggering an 'arginine switch' (Arg125 movement) that opens the primary hydrophobic pocket on ALG-2 to accommodate the PPYP motif of Alix; crystal structures of Ca2+-free, Ca2+-bound, and Alix peptide-complexed ALG-2 forms established this mechanism.","method":"X-ray crystallography; in vitro binding assay with ALG-2 and Alix mutants","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structures plus mutagenesis plus in vitro binding, single rigorous study","pmids":["18940611"],"is_preprint":false},{"year":2008,"finding":"Tsg101 acts as a positive regulator and Alix as a negative regulator of intraluminal vesicle budding into late endosomes, reconstituted in a novel quantitative biochemical in vitro budding assay; the process is ESCRT-dependent (inhibited by dominant-negative hVps4).","method":"In vitro reconstitution assay; dominant-negative VPS4; electron microscopy","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with specific inhibitors; multiple validation methods","pmids":["18768755"],"is_preprint":false},{"year":2008,"finding":"ALIX Bro1-CHMP4 and ALIX V-domain interactions are both required for cytokinetic abscission; additionally the C-terminus of Alix encodes a multimerization activity essential for Alix-dependent HIV-1 release and interaction with Tsg101.","method":"Dominant-negative and deletion mutagenesis; cytokinesis assay; HIV budding assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis with defined phenotypic readouts, single lab","pmids":["18641129"],"is_preprint":false},{"year":2009,"finding":"ALIX dimerizes via its middle V domain in solution; SAXS modeling shows dimeric ALIX adopts an elongated crescent shape; ALIX dimerization mutations prevent this and affect HIV-1 budding; dimeric ALIX bridges CHMP4B filaments formed in vitro, acting as a scaffold during membrane remodeling.","method":"Small-angle X-ray scattering (SAXS); dimerization mutagenesis; in vitro filament bridging assay; HIV budding assay","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 / Strong — SAXS structural modeling plus in vitro reconstitution plus functional validation","pmids":["19523902"],"is_preprint":false},{"year":2009,"finding":"ALG-2 bridges Alix and TSG101 as a Ca2+-dependent adaptor; the ALG-2-binding site on Alix (not the PSAP motif) is required for Ca2+-dependent pulldown of TSG101; dimeric ALG-2 is required for this bridging function.","method":"Strep-pulldown assays; ALG-2 knockdown; exogenous ALG-2 rescue; mutant analysis","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pull-down with knockdown and rescue, single lab, multiple methods","pmids":["19520058"],"is_preprint":false},{"year":2010,"finding":"The ALIX C-terminal proline-rich region (PRR) folds back against upstream domains causing auto-inhibition of V domain binding to viral late domains; mutations destabilizing this closed conformation open the V domain, increase ALIX membrane association, and enhance virus budding.","method":"Mutagenesis; in vitro binding assays; virus budding assays; membrane association assay","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis with functional budding assay and membrane fractionation, single lab","pmids":["21715492"],"is_preprint":false},{"year":2010,"finding":"ALIX contains an intramolecular interaction between Patch 2 of the Bro1 domain and the TSG101-docking site in the PRD that locks ALIX in a closed conformation, preventing CHMP4 and retroviral Gag protein binding; relieving this autoinhibition promotes ALIX interaction with partners and membrane association, and increases EIAV budding.","method":"ALIX truncation/mutation analysis; co-IP; membrane fractionation; EIAV budding assay","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis with co-IP and functional assay, single lab","pmids":["20929444"],"is_preprint":false},{"year":2010,"finding":"Nedd4-1 binds ALIX directly (co-IP and yeast two-hybrid); Nedd4-1 is required for ALIX ubiquitination; ALIX recruits Nedd4-1 through the LYPX(n)L motif to facilitate HIV-1 release; Nedd4-1 catalytic activity is required for this function.","method":"Co-immunoprecipitation; yeast two-hybrid; siRNA knockdown; HIV budding assay; catalytic mutant","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP plus siRNA plus functional assay, single lab","pmids":["20519395"],"is_preprint":false},{"year":2012,"finding":"ALIX binds a YPX(3)L motif in the GPCR PAR1 via its central V domain to mediate ubiquitin-independent, ESCRT-III-dependent MVB/lysosomal sorting of PAR1; depletion of ALIX impairs PAR1 sorting into ILVs and lysosomal degradation.","method":"siRNA depletion; site-directed mutagenesis of YPX(3)L; co-immunoprecipitation; MVB sorting assay","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA plus mutagenesis plus sorting assay, single lab","pmids":["22547407"],"is_preprint":false},{"year":2012,"finding":"The V domain of ALIX binds directly and selectively to K63-linked polyubiquitin chains (preference for chains >3 Ub); mutations in two putative Ub-binding sites on a helical surface of the V domain inhibit polyUb binding in vitro and impair lentiviral budding in cells.","method":"Affinity capture/mass spectrometry; in vitro binding assays; mutagenesis; lentiviral budding assay","journal":"Developmental cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro binding plus mutagenesis plus functional assay, single lab","pmids":["23201121"],"is_preprint":false},{"year":2013,"finding":"Yeast Bro1 (Alix ortholog) functions as a ubiquitin receptor via the N-terminal trihelical arm of its V domain; Bro1 binds ubiquitin and sorts ubiquitinated cargo in parallel with ESCRT-0, demonstrated by structural studies, mutagenesis, and synthetic genetic interactions with ESCRT-0.","method":"Multiple structural approaches (X-ray/NMR); mutagenesis; genetic epistasis (synthetic lethality screen)","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — structure plus mutagenesis plus genetic epistasis, single comprehensive study","pmids":["23726974"],"is_preprint":false},{"year":2014,"finding":"The syndecan-syntenin-ALIX pathway controls exosome biogenesis and ILV budding; ARF6 and its effector PLD2 regulate syntenin exosome production by controlling ILV budding into MVBs in an ALIX-dependent manner; this pathway does not affect HIV-1 budding.","method":"siRNA depletion; overexpression; electron microscopy; exosome isolation","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA with multiple readouts, single lab","pmids":["24637612"],"is_preprint":false},{"year":2015,"finding":"ATG12-ATG3 conjugate interacts with Alix (PDCD6IP) and controls multiple Alix-dependent processes including late endosome distribution, exosome biogenesis, and viral budding; Alix is functionally required for efficient basal (but not starvation-induced) autophagy.","method":"Co-immunoprecipitation; siRNA/knockout; autophagy flux assay; exosome isolation; electron microscopy","journal":"Nature cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus KO with defined phenotypic readouts, single lab","pmids":["25686249"],"is_preprint":false},{"year":2015,"finding":"Heparanase stimulates syndecan-syntenin-ALIX-dependent exosome production by trimming heparan sulfate chains on syndecans, promoting intraluminal budding of syndecan and syntenin-1 in an ALIX-dependent manner.","method":"siRNA depletion; heparanase treatment; exosome isolation and quantification; electron microscopy","journal":"Cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA with multiple readouts plus biochemical rescue, single lab","pmids":["25732677"],"is_preprint":false},{"year":2015,"finding":"ALG-2 calcium-dependent interaction with Alix relieves ALIX intramolecular autoinhibition, promoting CHMP4-dependent ALIX membrane association and MVB sorting of activated EGFR; this ALG-2 activation of ALIX is specific to MVB sorting and does not affect cytokinetic abscission or EIAV budding.","method":"Co-immunoprecipitation; membrane fractionation; MVB sorting assay; siRNA; mutagenesis","journal":"Cell discovery","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus functional sorting assay plus pathway specificity demonstration, single lab","pmids":["27462417"],"is_preprint":false},{"year":2015,"finding":"ARRDC3 (α-arrestin) regulates ALIX ubiquitination by WWP2 E3 ligase; ARRDC3 and WWP2 are required for ALIX interaction with activated PAR1 and CHMP4B, and for PAR1 lysosomal sorting; WWP2 interacts with ARRDC3 (not ALIX) to ubiquitinate ALIX.","method":"siRNA screen; co-immunoprecipitation; ubiquitination assay; GPCR sorting assay","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA plus co-IP plus ubiquitination assay, single lab","pmids":["26490116"],"is_preprint":false},{"year":2015,"finding":"ALIX is required for abscission completion in Drosophila female germline stem cells (fGSCs) in vivo; ALIX and ESCRT-III component Shrub interact directly, co-localize at midbody rings, and their direct interaction is required for normal abscission kinetics.","method":"Drosophila loss-of-function genetics; co-immunoprecipitation; fluorescence microscopy in vivo","journal":"PLoS genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo genetic loss-of-function with co-IP and localization, single lab","pmids":["25635693"],"is_preprint":false},{"year":2016,"finding":"ALIX and ESCRT-I/II function as parallel ESCRT-III recruiters during cytokinetic abscission; ALIX specifically recruits CHMP4C (abscission checkpoint component) while ESCRT-II/CHMP6 cooperate with ESCRT-I to recruit CHMP4B; ALIX depletion causes furrow regression in cells with chromosome bridges, implicating ALIX in abscission checkpoint signaling.","method":"siRNA depletion; fluorescence microscopy; cytokinesis/abscission assay; epistasis analysis","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA epistasis with defined phenotypic readouts, single lab","pmids":["26929449"],"is_preprint":false},{"year":2016,"finding":"ALIX mediates ubiquitin-independent lysosomal sorting of the P2Y1 GPCR via a YPX3L motif in the second intracellular loop; site-directed mutation of this motif or ALIX depletion inhibits P2Y1 MVB/lysosomal sorting.","method":"siRNA depletion; site-directed mutagenesis; MVB sorting/lysosomal degradation assay","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA plus mutagenesis with functional readout, single lab","pmids":["27301021"],"is_preprint":false},{"year":2016,"finding":"Alix binds Ago2 (Argonaute 2) by co-immunoprecipitation; ALIX knockdown decreases miRNA content in extracellular vesicles without reducing vesicle number, indicating a role for Alix in miRNA packaging into EVs.","method":"Co-immunoprecipitation; siRNA knockdown; RT-qPCR for miRNAs in EVs","journal":"International journal of molecular medicine","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single co-IP and siRNA experiment, single lab, single method per claim","pmids":["26935291"],"is_preprint":false},{"year":2016,"finding":"Alix is required for clathrin-independent endocytosis (CIE) but not clathrin-mediated endocytosis; in Alix knockout cells, fluid-phase endocytosis and CIE cargoes (cholera toxin, IL-2 receptor) are impaired; Alix acts with endophilin-A to promote CIE and regulate cell migration.","method":"Alix knockout cells; endocytosis assays; signaling assays; cell migration assay","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO with defined endocytic phenotype and multiple cargoes tested, single lab","pmids":["27244115"],"is_preprint":false},{"year":2016,"finding":"Alix interacts with F-actin, the Par complex, and ZO-1 to assemble the apically restricted actomyosin-tight junction complex in epithelial cells; Alix knockout mice develop hydrocephalus due to defects in epithelial polarity, tight junction formation, and excessive cell extrusion in choroid plexus/ependyma.","method":"Knockout mouse model; co-immunoprecipitation; fluorescence/electron microscopy; phenotypic analysis","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse with co-IP and defined structural phenotype, single lab","pmids":["27336173"],"is_preprint":false},{"year":2018,"finding":"Alix undergoes S-palmitoylation as a post-translational modification; this lipid modification influences Alix protein-protein interaction with CD9 (tetraspanin) and affects the structural organization of the lipid bilayer of extracellular vesicles.","method":"Palmitoylation assay (acyl-RAC); co-immunoprecipitation; biophysical lipid bilayer analysis","journal":"Biochimica et biophysica acta. General subjects","confidence":"Low","confidence_rationale":"Tier 3 / Weak — palmitoylation assay plus co-IP, single lab, limited mechanistic depth","pmids":["30251702"],"is_preprint":false},{"year":2018,"finding":"ALIX depletion results in prolonged EGFR activity after stimulation, defective PD-L1 trafficking through MVBs, reduced PD-L1 exosomal secretion, and redistribution of PD-L1 to the cell surface, conferring an EGFR-dependent immunosuppressive phenotype.","method":"siRNA depletion; EGFR signaling assays; flow cytometry for PD-L1; exosome isolation; mouse tumor model","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA with multiple readouts including in vivo model, single lab","pmids":["30021161"],"is_preprint":false},{"year":2019,"finding":"In Drosophila, ALIX is recruited to the midbody via its V domain through direct interaction with an LxxLF motif in centralspindlin component Pavarotti (MKLP1), using the same conserved hydrophobic V-domain pocket that binds LYPXnL virus proteins; this mechanism is CEP55-independent.","method":"Co-immunoprecipitation; domain mapping/mutagenesis; fluorescence microscopy; Drosophila genetics","journal":"Current biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP with domain mutagenesis and in vivo localization, single lab","pmids":["31607533"],"is_preprint":false},{"year":2019,"finding":"Phosphorylated MLKL (necroptosis mediator) is removed from membranes via ALIX-syntenin-1-mediated exocytosis; ALIX and syntenin-1 are detected in membrane-localized MLKL immunoprecipitates by protein cross-linking/affinity purification.","method":"Protein cross-linking; affinity purification; functional exocytosis assay","journal":"Science signaling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — crosslinking-AP plus functional assay, single lab","pmids":["31138766"],"is_preprint":false},{"year":2020,"finding":"ALIX (active form) recruits ESCRT-III proteins to endosomes independently of other ESCRTs but requiring lysobisphosphatidic acid (LBPA) in vivo; this can be reconstituted on supported bilayers in vitro; this ALIX-ESCRT-III pathway promotes sorting of tetraspanins specifically to exosomes.","method":"In vitro reconstitution on supported bilayers; ALIX activation mutants; siRNA; electron microscopy","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution on bilayers plus in vivo siRNA with cargo-specific readout, single comprehensive study","pmids":["32049272"],"is_preprint":false},{"year":2020,"finding":"The ESCRT-III machinery localizes to the abscission site via a plasma membrane-to-ESCRT module composed of syndecan-4 (transmembrane proteoglycan), ALIX, and syntenin; direct interactions between ALIX, syntenin, and syndecan-4 are essential for ESCRT-III enrichment at the abscission site (but not at the midbody); depletion of any of the three proteins delays abscission.","method":"Proteomics of isolated midbodies (Flemmingsome); siRNA depletion; co-immunoprecipitation; fluorescence microscopy","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus siRNA with defined abscission phenotype plus proteomic identification, single lab","pmids":["32321914"],"is_preprint":false},{"year":2020,"finding":"ALIX and Syntenin-1 are required for proper subcellular localization and efficient EV secretion of APP; their knockdown sequesters APP in ER and endolysosomal compartments respectively; this pathway is ESCRT-independent.","method":"Inducible siRNA knockdown; subcellular localization; EV isolation and analysis","journal":"BMC molecular and cell biology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — siRNA with localization readout, single lab, indirect mechanistic evidence","pmids":["32731849"],"is_preprint":false},{"year":2020,"finding":"The ALIX proline-rich domain (PRD) contains three tandem proline-rich motifs that compete for a single binding site on TSG101-UEV; the C-terminal tyrosine-rich portion of ALIX-PRD forms amyloid fibrils that dissolve upon hyperphosphorylation by Src kinase, revealing a phosphorylation-controlled reversible amyloid mechanism regulating ALIX function.","method":"Heteronuclear NMR spectroscopy; relaxation dispersion; thioflavin T/Congo red assays; electron microscopy; Src kinase phosphorylation","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — NMR with kinetic measurements, in vitro reconstitution of amyloid formation/dissolution, mutagenesis, multiple orthogonal methods","pmids":["32917811"],"is_preprint":false},{"year":2021,"finding":"ALIX (not the broader ESCRT machinery) is required specifically for apical exosome release in polarized epithelial cells via the ALIX-Syntenin1-Syndecan1 machinery, while basolateral exosome release depends on ceramide; these represent two independent polarized exosome release pathways.","method":"siRNA depletion; polarized epithelial cell model; exosome isolation; directional secretion assay","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA with directional exosome assay in polarized cells, single lab","pmids":["33724661"],"is_preprint":false},{"year":2012,"finding":"Alix is a substrate of the muscle-specific ubiquitin E3 ligase Ozz-E3; Ozz interaction with Alix changes Alix conformation and promotes its ubiquitination, regulating Alix levels at cortactin-containing compartments; Alix knockdown in muscle cells affects F-actin amount and distribution, impairs sarcolemmal protrusion formation and cell motility.","method":"Co-immunoprecipitation (endogenous proteins); ubiquitination assay; Ozz knockout mouse; siRNA knockdown; actin/morphology assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — endogenous co-IP plus KO mouse plus siRNA with functional readout, single lab","pmids":["22334701"],"is_preprint":false},{"year":2023,"finding":"SUMOylated hnRNPA2B1 (SUMO2 at K108) is recognized by the SUMO interaction motif (SIM) of ALIX, activating ALIX to recruit ESCRT-III for sorting of circTLCD4-RWDD3 into extracellular vesicles; mutating the SIM in ALIX or K108 in hnRNPA2B1 blocks EV packaging and inhibits lymphatic metastasis in vivo.","method":"Co-immunoprecipitation; SUMOylation assay; SIM mutation in ALIX; in vivo tumor model","journal":"Signal transduction and targeted therapy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP with mutagenesis plus in vivo validation, single lab","pmids":["37925421"],"is_preprint":false}],"current_model":"PDCD6IP/Alix is a multifunctional ESCRT-associated adaptor protein that operates as a conformationally regulated scaffold: its N-terminal Bro1 domain binds CHMP4/ESCRT-III subunits (using a hydrophobic code distinct from MIT-domain partners), its central V domain binds YPX(n)L late-domain motifs of retroviruses and cellular receptors (including GPCRs via YPX(n)L sequences) as well as K63-linked polyubiquitin chains, and its C-terminal proline-rich domain (PRD) engages ALG-2 (in a Ca2+-dependent arginine-switch mechanism), TSG101, endophilins, and CEP55/centralspindlin; the PRD also folds back onto the Bro1/V domains to maintain an autoinhibited closed conformation that is relieved by Ca2+/ALG-2, membrane recruitment, partner binding, or Ozz/Nedd4/WWP2-mediated ubiquitination, thereby activating ALIX to recruit ESCRT-III and drive membrane fission during enveloped virus budding, MVB intraluminal vesicle formation (including a tetraspanin-specific pathway requiring LBPA), exosome biogenesis (via syndecan-syntenin-ALIX and ARF6-PLD2 axes), cytokinetic abscission (recruited by CEP55 in mammals or centralspindlin in Drosophila to the midbody), clathrin-independent endocytosis, and actin cytoskeleton organization."},"narrative":{"mechanistic_narrative":"PDCD6IP/Alix is a conformationally regulated ESCRT-associated scaffold that links membrane cargo and accessory proteins to the ESCRT-III machinery to drive membrane fission across multiple cellular processes [PMID:17350572, PMID:32049272]. Its architecture comprises an N-terminal Bro1 domain that binds CHMP4/ESCRT-III subunits through a hydrophobic code distinct from the MIT-domain partners of VPS4 [PMID:18511562], a central V domain that recognizes YPX(n)L late-domain motifs of retroviral Gag proteins and cellular GPCRs as well as K63-linked polyubiquitin chains [PMID:17350572, PMID:17277784, PMID:18066081, PMID:22547407, PMID:23201121], and a C-terminal proline-rich domain that engages ALG-2 in a Ca2+-dependent arginine-switch manner, TSG101, and endophilins [PMID:12034747, PMID:14999017, PMID:18940611, PMID:32917811]. The proline-rich region folds back onto the Bro1/V domains to lock Alix in an autoinhibited closed conformation that is relieved by Ca2+/ALG-2, membrane recruitment, partner binding, or E3-ligase-mediated ubiquitination, thereby licensing CHMP4 recruitment and membrane association [PMID:21715492, PMID:20929444, PMID:27462417, PMID:22334701]. Through these interactions Alix mediates enveloped virus budding [PMID:14505569, PMID:17350572], multivesicular body intraluminal vesicle formation and a tetraspanin-specific exosome pathway requiring lysobisphosphatidic acid [PMID:18768755, PMID:32049272], syndecan-syntenin-driven exosome biogenesis [PMID:24637612, PMID:25732677], ubiquitin-independent MVB/lysosomal sorting of GPCRs via YPX(n)L motifs [PMID:22547407, PMID:27301021], and cytokinetic abscission, where it is recruited to the midbody by CEP55 in mammals or centralspindlin in Drosophila and delivers CHMP4 to the abscission site [PMID:17853893, PMID:26929449, PMID:31607533]. Alix additionally organizes the actin cytoskeleton and epithelial junctions; its loss in mice produces hydrocephalus from defects in epithelial polarity and tight-junction assembly [PMID:16966331, PMID:27336173], and it supports clathrin-independent endocytosis together with endophilin-A [PMID:27244115].","teleology":[{"year":1999,"claim":"Established Alix's founding biochemical partnership, defining it as a Ca2+-regulated effector and hinting at a role in regulated death/signaling.","evidence":"Yeast two-hybrid and co-IP identifying ALG-2 as a calcium-dependent partner","pmids":["10200558"],"confidence":"Medium","gaps":["Functional consequence of the ALG-2 interaction unresolved at this stage","No structural basis for Ca2+ dependence yet","No link to membrane trafficking established"]},{"year":2002,"claim":"Mapped the proline-rich domain as a protein-interaction hub by identifying endophilin binding, linking Alix to membrane-deforming machinery.","evidence":"Yeast two-hybrid, co-IP and overlay assays with PXRPPPP consensus mapping","pmids":["12034747"],"confidence":"Medium","gaps":["Physiological process served by Alix-endophilin binding undefined","No structural detail of the interface"]},{"year":2003,"claim":"Placed Alix in the ESCRT pathway by showing it bridges retroviral Gag late domains to ESCRT-III and TSG101, defining its role in viral budding.","evidence":"Co-IP, binding and functional budding assays with HIV-1 p6, EIAV p9, and CHMP4b","pmids":["14505569","12860994","12588984"],"confidence":"High","gaps":["Atomic basis of YPX(n)L and CHMP4 recognition not yet known","How budding is spatially coordinated unresolved"]},{"year":2004,"claim":"Defined the ALG-2 binding site on Alix and uncovered counter-roles in receptor downregulation, endosome organization, and non-apoptotic death, broadening Alix beyond budding.","evidence":"Mutagenesis/overlay/co-IP mapping ALG-2 to residues 794-827; siRNA and ubiquitination assays for EGFR/Cbl; Src kinase assays; paraptosis assays","pmids":["14999017","15456872","15557335","15195070","15086793"],"confidence":"Medium","gaps":["Mechanistic link between EGFR regulation and ESCRT sorting unclear","Src phosphorylation site and structural effect not resolved at this stage"]},{"year":2005,"claim":"Connected Alix to endosome positioning and the lipid environment of MVBs, distinguishing its role from EGFR degradation and recycling.","evidence":"RNAi with immunofluorescence and immunoelectron microscopy measuring LBPA and endosome distribution","pmids":["15914539"],"confidence":"Medium","gaps":["Molecular basis of LBPA dependence undefined","Mechanism linking Alix loss to aberrant actin/clathrin structures unclear"]},{"year":2006,"claim":"Demonstrated direct actin-cytoskeleton engagement, establishing a structural role for Alix beyond membrane scission.","evidence":"Cell-free direct binding to F-actin, cortactin, alpha-actinin; siRNA phenotypes on stress fibers","pmids":["16966331"],"confidence":"Medium","gaps":["How cytoskeletal and ESCRT functions are coordinated unknown","In vivo relevance not addressed here"]},{"year":2007,"claim":"Solved the structural logic of Alix: defined Bro1 and V domain architecture, the YPX(n)L pocket, CHMP4 binding, and the CEP55-dependent midbody recruitment for abscission.","evidence":"Crystallography of Bro1/V domains and V-domain:late-domain complexes; proteomics, co-IP, siRNA and cytokinesis assays for CEP55/TSG101; NC-Bro1 packaging","pmids":["17350572","17277784","18066081","17853893","18032513"],"confidence":"High","gaps":["Conformational regulation of these interactions not yet defined","Cargo-specific pathway selection unresolved"]},{"year":2008,"claim":"Defined the molecular grammar of partner selection and oligomeric scaffolding: the CHMP4 hydrophobic code, the ALG-2 arginine switch, V-domain dimerization, and a positive/negative regulatory split with TSG101 in ILV budding.","evidence":"Crystallography of Bro1:CHMP4 and Ca2+/ALG-2 states; SAXS dimer modeling and filament bridging; in vitro ILV reconstitution; abscission/HIV mutagenesis","pmids":["18511562","18940611","18768755","18641129","19523902"],"confidence":"High","gaps":["How dimerization is triggered in cells unclear","Reconciliation of Alix as negative regulator in vitro vs positive elsewhere"]},{"year":2010,"claim":"Identified intramolecular autoinhibition as the master regulatory principle, with the PRD folding back to occlude V and Bro1 surfaces and ubiquitination/E3 ligases as activating inputs.","evidence":"Truncation/mutagenesis with co-IP, membrane fractionation, budding assays; Nedd4-1 and ALG-2 bridging studies","pmids":["21715492","20929444","20519395","19520058"],"confidence":"Medium","gaps":["Quantitative dynamics of open/closed equilibrium not measured","Cell-type-specific activation inputs incompletely mapped"]},{"year":2012,"claim":"Extended Alix cargo recognition to ubiquitin-independent GPCR sorting via YPX(3)L motifs and to direct K63-polyubiquitin binding, and identified Ozz-E3 ubiquitination as a conformational/abundance regulator.","evidence":"siRNA/mutagenesis PAR1 sorting; in vitro polyUb binding and mutagenesis; endogenous co-IP, Ozz KO mouse and actin phenotypes","pmids":["22547407","23201121","22334701"],"confidence":"Medium","gaps":["Relative contributions of ubiquitin-dependent vs -independent cargo capture unclear","Tissue-specific roles of Ozz regulation undefined"]},{"year":2013,"claim":"Established the ancestral V-domain ubiquitin-receptor function via the yeast Bro1 ortholog, paralleling ESCRT-0 in cargo sorting.","evidence":"Structural studies, mutagenesis, and synthetic genetic epistasis with ESCRT-0 in yeast","pmids":["23726974"],"confidence":"High","gaps":["Conservation of this ubiquitin-receptor role in human Alix not formally proven here","Interplay with V-domain YPX(n)L binding unresolved"]},{"year":2014,"claim":"Defined the syndecan-syntenin-ALIX exosome pathway and its ARF6-PLD2 control, establishing a budding route distinct from viral budding.","evidence":"siRNA, overexpression, EM and exosome isolation","pmids":["24637612"],"confidence":"Medium","gaps":["How ARF6-PLD2 lipids feed into Alix activation unclear","Cargo selectivity of this pathway incompletely defined"]},{"year":2015,"claim":"Integrated Alix into autophagy, basal endosomal exosome biogenesis, and demonstrated pathway-selective activation, while resolving exosome production regulators including heparanase and parallel abscission recruiters.","evidence":"ATG12-ATG3 co-IP/KO; ALG-2 activation specific to EGFR MVB sorting; heparanase exosome assays; Drosophila Shrub genetics; CHMP4C-specific abscission siRNA epistasis; ARRDC3/WWP2 ubiquitination","pmids":["25686249","27462417","25732677","25635693","26929449","26490116"],"confidence":"Medium","gaps":["How a single scaffold achieves pathway-specific activation mechanistically unclear","Crosstalk between activating inputs (ALG-2, ubiquitination, lipids) unresolved"]},{"year":2016,"claim":"Broadened Alix's cellular roles to clathrin-independent endocytosis, epithelial junction/actin assembly with an in vivo hydrocephalus phenotype, additional GPCR sorting, and EV miRNA packaging.","evidence":"Alix KO endocytosis assays with endophilin-A; KO mouse epithelial polarity/tight-junction analysis; P2Y1 YPX3L sorting; Ago2 co-IP and EV miRNA quantification","pmids":["27244115","27336173","27301021","26935291"],"confidence":"Medium","gaps":["Mechanistic link between Alix's actin-junction role and its ESCRT scaffold function unclear","Ago2/miRNA-packaging mechanism rests on a single low-confidence study"]},{"year":2018,"claim":"Linked Alix-dependent MVB sorting to immune regulation through PD-L1 trafficking and EV secretion, and identified S-palmitoylation as a lipid-modification input.","evidence":"siRNA, flow cytometry, exosome isolation and mouse tumor model for PD-L1; acyl-RAC palmitoylation and CD9 co-IP","pmids":["30021161","30251702"],"confidence":"Medium","gaps":["Palmitoylation site and functional requirement rest on a single low-confidence study","How Alix selects PD-L1 cargo unresolved"]},{"year":2019,"claim":"Revealed an evolutionarily flexible recruitment logic — the V-domain LYPX(n)L pocket also captures the centralspindlin LxxLF motif for CEP55-independent midbody recruitment — and removal of phospho-MLKL via Alix-syntenin exocytosis.","evidence":"Co-IP, domain mutagenesis and Drosophila genetics for Pavarotti; cross-linking/AP and exocytosis assay for MLKL","pmids":["31607533","31138766"],"confidence":"Medium","gaps":["Whether mammalian centralspindlin uses the same pocket unresolved","Regulation switching between viral and midbody ligands at the same pocket unclear"]},{"year":2020,"claim":"Reconstituted active Alix recruiting ESCRT-III on LBPA-containing bilayers for tetraspanin exosome sorting, defined the syndecan-4-syntenin-Alix module at the abscission site, and resolved a phosphorylation-controlled reversible amyloid in the PRD that regulates TSG101 competition.","evidence":"In vitro bilayer reconstitution and siRNA/EM for tetraspanins; Flemmingsome proteomics and abscission siRNA; NMR/relaxation/ThT and Src phosphorylation of the PRD","pmids":["32049272","32321914","32917811","32731849","32049272"],"confidence":"High","gaps":["Physiological trigger of PRD amyloid formation in cells unclear","How the syndecan-4 module is distinguished from midbody recruitment incompletely defined"]},{"year":2021,"claim":"Showed Alix governs a polarity-specific exosome route, mediating apical exosome release distinct from ceramide-dependent basolateral release.","evidence":"siRNA and directional exosome assays in polarized epithelial cells","pmids":["33724661"],"confidence":"Medium","gaps":["Molecular basis of apical vs basolateral sorting unresolved","Link to the epithelial junction role of Alix not connected"]},{"year":2023,"claim":"Identified a SUMO-dependent cargo-capture mode in which the Alix SIM recognizes SUMOylated hnRNPA2B1 to drive circRNA packaging into EVs with pro-metastatic consequences.","evidence":"Co-IP, SUMOylation assays, SIM mutation and in vivo metastasis model","pmids":["37925421"],"confidence":"Medium","gaps":["Generality of the SIM-dependent cargo route beyond this circRNA unknown","How SUMO recognition integrates with autoinhibition release unclear"]},{"year":null,"claim":"How a single autoinhibited scaffold integrates Ca2+/ALG-2, ubiquitination, SUMO, lipid, and phosphorylation/amyloid inputs to select among virus budding, ILV/exosome biogenesis, abscission, endocytosis, and cytoskeletal roles in a given cell remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified quantitative model of conformational switching across pathways","Cargo- and pathway-specific activation inputs only partly mapped","Mammalian counterpart of several Drosophila/yeast findings not formally confirmed"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[2,12,13,17,21]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[11,38,48]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[20,43]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[7,18]}],"localization":[{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[3,10,25,31]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[22,38,44]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[9,10]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[11,48]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[3,18,28,43]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[13,34,44]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[2,12,14]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[7,25,35]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[36,49]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[29]}],"complexes":["ESCRT-III (CHMP4)","syndecan-syntenin-ALIX module","actomyosin-tight junction complex"],"partners":["PDCD6 (ALG-2)","CHMP4B","TSG101","CEP55","SDCBP (SYNTENIN)","ENDOPHILIN","NEDD4","PAR1"],"other_free_text":[]}},"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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soluble vacuolar proteins.","date":"2022","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/35533279","citation_count":26,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":47424,"output_tokens":11098,"usd":0.154371,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":21751,"output_tokens":6410,"usd":0.134502,"stage2_stop_reason":"end_turn"},"total_usd":0.288873,"stage1_batch_id":"msgbatch_013a8chtv3AWyTqv4z4sEF4C","stage2_batch_id":"msgbatch_01AHsuW63vinsvuTgzmQ3n62","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1999,\n      \"finding\": \"Alix (PDCD6IP) was identified as a novel binding partner of ALG-2 (apoptosis-linked gene 2) in a calcium-dependent manner, established by yeast two-hybrid screening and co-immunoprecipitation experiments.\",\n      \"method\": \"Yeast two-hybrid screen; co-immunoprecipitation\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP confirming yeast two-hybrid, single lab, two orthogonal methods\",\n      \"pmids\": [\"10200558\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Alix binds endophilins (SH3p4, SH3p8, SH3p13) through a proline-rich domain interaction (PXRPPPP consensus) at its C-terminal half, demonstrated by yeast two-hybrid, co-immunoprecipitation, and overlay experiments; overexpression of Alix-CT causes cytoplasmic vacuolization enhanced by endophilin co-expression.\",\n      \"method\": \"Yeast two-hybrid; co-immunoprecipitation; overlay assay; cell transfection/overexpression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP and overlay assays, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"12034747\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"HIV-1 Gag p6 contains a second L-domain region that binds AIP1/Alix (PDCD6IP); Alix also interacts with Tsg101 and ESCRT-III homologs, and binds the EIAV p9 L domain, identifying Alix as a component of the viral budding machinery linking retroviral Gag proteins to ESCRT-III.\",\n      \"method\": \"Co-immunoprecipitation; binding assays; functional budding assays\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP with multiple partners, functional validation of budding, independently replicated across labs\",\n      \"pmids\": [\"14505569\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Alix interacts with CHMP4b (human Snf7 homolog) as demonstrated by GST pull-down and co-immunoprecipitation; CHMP4b and Alix co-localize at perinuclear puncta overlapping with endosomal markers, and cooperate in multivesicular body formation.\",\n      \"method\": \"Yeast two-hybrid; GST pull-down; co-immunoprecipitation; fluorescence microscopy\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro pull-down plus co-IP plus localization, replicated by multiple labs\",\n      \"pmids\": [\"12860994\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Both fungal PalA and mammalian AIP1/Alix recognize the YPXL/I protein-protein binding motif, where Tyr, Pro, and Leu/Ile are crucial for binding, established by mutagenesis of the motif in PacC substrates and binding assays.\",\n      \"method\": \"Binding assays; site-directed mutagenesis of YPXL/I motif\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis plus binding assay, single lab\",\n      \"pmids\": [\"12588984\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Paraptosis (non-apoptotic cell death induced by IGFIR) is inhibited specifically by AIP-1/Alix overexpression; this inhibition does not affect apoptosis and is MAPK/JNK-1-mediated, placing Alix as a negative regulator of IGFIR-induced paraptosis.\",\n      \"method\": \"Cell transfection/overexpression; antisense oligonucleotides; MEK-2 inhibitors; cell death assays\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — functional knockdown/overexpression with specific phenotypic readout, single lab\",\n      \"pmids\": [\"15195070\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"ALG-2 interacts with the C-terminal proline-rich region of Alix (residues 794–827) through four tandem PxY repeats in a Ca2+-dependent manner; specific proline and tyrosine residues within this region are critical for binding affinity, demonstrated by yeast two-hybrid, overlay assay, and co-immunoprecipitation with deletion/alanine-substitution mutants.\",\n      \"method\": \"Yeast two-hybrid; biotin-tagged overlay assay; co-immunoprecipitation; alanine mutagenesis\",\n      \"journal\": \"Journal of biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — mutagenesis combined with co-IP and overlay, multiple methods, defines binding region precisely\",\n      \"pmids\": [\"14999017\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Alix/AIP1 antagonizes EGFR downregulation by attenuating the Cbl-SETA/CIN85 interaction and inhibiting Cbl-mediated ubiquitination of EGFR; siRNA knockdown of Alix promotes EGFR internalization and downregulation.\",\n      \"method\": \"Co-immunoprecipitation; siRNA knockdown; ubiquitination assays; receptor internalization assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA plus co-IP plus ubiquitination assay, single lab, multiple methods\",\n      \"pmids\": [\"15456872\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Alix co-immunoprecipitates with exosomal transferrin receptor, and a peptide (P1, YTRFSLARQV) from the TfR cytosolic domain binds both hsc70 and Alix (via homology to its fungal homolog PalA), suggesting Alix contributes to exosomal sorting of TfR via the YTRF motif.\",\n      \"method\": \"Co-immunoprecipitation; peptide binding/affinity chromatography\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single co-IP and peptide binding, indirect evidence for YTRF interaction\",\n      \"pmids\": [\"15086793\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Src phosphorylates Alix at its C-terminal tyrosine-rich region; this phosphorylation causes Alix to translocate from membrane/cytoskeleton to cytoplasm and reduces its interactions with SETA/CIN85, EGFR, and Pyk2, antagonizing Alix's negative regulation of receptor internalization and cell adhesion.\",\n      \"method\": \"In vitro kinase assay; co-immunoprecipitation; subcellular fractionation; siRNA/overexpression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro kinase assay plus co-IP plus localization, single lab\",\n      \"pmids\": [\"15557335\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Alix depletion by RNAi causes redistribution of early endosomes from peripheral to perinuclear location, accumulation of unusual actin structures containing clathrin and cortactin, and reduction of lysobisphosphatidic acid levels in multivesicular endosomes, without affecting transferrin recycling or EGF receptor degradation.\",\n      \"method\": \"RNAi knockdown; immunofluorescence microscopy; quantitative immunoelectron microscopy\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA with defined phenotypic readouts, multiple imaging methods, single lab\",\n      \"pmids\": [\"15914539\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Alix directly interacts with F-actin (via both Bro1 and proline-rich domains), cortactin (via the N-terminal half of the middle region), and alpha-actinin (via the C-terminal half of the middle region) in cell-free systems; Alix knockdown decreases F-actin content and inhibits stress fiber assembly and lamellipodial localization of cortactin.\",\n      \"method\": \"In vitro direct binding (cell-free); co-immunoprecipitation; siRNA knockdown; fluorescence microscopy\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro direct binding assay plus siRNA functional phenotype, single lab\",\n      \"pmids\": [\"16966331\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Crystal structures of ALIX reveal an N-terminal Bro1 domain and a central V domain composed of two three-helix bundle arms; YPX(n)L late domains 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; functional budding assays\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with mutagenesis and functional validation in a single rigorous study\",\n      \"pmids\": [\"17350572\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"ALIX and TSG101/ESCRT-I are recruited to midbodies of dividing cells through direct interactions between CEP55 and GPP-based motifs within TSG101 and ALIX; ALIX point mutants blocking CEP55 and CHMP4/ESCRT-III binding inhibit cytokinetic abscission; depletion of ALIX inhibits the abscission step of HeLa cell cytokinesis.\",\n      \"method\": \"Proteomics; co-immunoprecipitation; siRNA depletion; fluorescence microscopy; cytokinesis assay\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, siRNA depletion with defined abscission phenotype, mutagenesis, replicated by other labs\",\n      \"pmids\": [\"17853893\"],\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 reveal that both bind the same hydrophobic pocket but adopt different conformations; binding affinity and budding function require key interface residues identified by mutagenesis.\",\n      \"method\": \"X-ray crystallography; surface plasmon resonance; mutagenesis; functional budding assays\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structures with mutagenesis and biophysical binding measurements in one rigorous study\",\n      \"pmids\": [\"17277784\", \"18066081\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"ALIX also engages HIV-1 Gag through the nucleocapsid (NC) domain via the Bro1 domain (in addition to the V domain/p6 interaction); this NC-Bro1 interaction depends on NC zinc fingers and allows ALIX packaging into viral particles; zinc finger mutations affect particle production similarly to PTAP deletion.\",\n      \"method\": \"Co-immunoprecipitation; viral packaging assays; nuclease treatment; mutagenesis\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP with mutagenesis and functional assay, single lab\",\n      \"pmids\": [\"18032513\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Crystal structures of ALIX Bro1 domain in complex with C-terminal peptides of CHMP4A-C show that CHMP4 C-terminal peptides form amphipathic helices binding across the concave surface of Bro1; a hydrophobic code (M/L/IxxLxxW) distinguishes CHMP4 binding to Bro1-domain proteins from CHMP1-3 binding to MIT-domain proteins (VPS4); mutations in CHMP4 recognition residues block ALIX-dependent HIV-1 budding.\",\n      \"method\": \"X-ray crystallography; mutagenesis; HIV-1 budding assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with mutagenesis and functional validation in one rigorous study\",\n      \"pmids\": [\"18511562\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"ALG-2 binds Ca2+ through EF3, triggering an 'arginine switch' (Arg125 movement) that opens the primary hydrophobic pocket on ALG-2 to accommodate the PPYP motif of Alix; crystal structures of Ca2+-free, Ca2+-bound, and Alix peptide-complexed ALG-2 forms established this mechanism.\",\n      \"method\": \"X-ray crystallography; in vitro binding assay with ALG-2 and Alix mutants\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structures plus mutagenesis plus in vitro binding, single rigorous study\",\n      \"pmids\": [\"18940611\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Tsg101 acts as a positive regulator and Alix as a negative regulator of intraluminal vesicle budding into late endosomes, reconstituted in a novel quantitative biochemical in vitro budding assay; the process is ESCRT-dependent (inhibited by dominant-negative hVps4).\",\n      \"method\": \"In vitro reconstitution assay; dominant-negative VPS4; electron microscopy\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with specific inhibitors; multiple validation methods\",\n      \"pmids\": [\"18768755\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"ALIX Bro1-CHMP4 and ALIX V-domain interactions are both required for cytokinetic abscission; additionally the C-terminus of Alix encodes a multimerization activity essential for Alix-dependent HIV-1 release and interaction with Tsg101.\",\n      \"method\": \"Dominant-negative and deletion mutagenesis; cytokinesis assay; HIV budding assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis with defined phenotypic readouts, single lab\",\n      \"pmids\": [\"18641129\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"ALIX dimerizes via its middle V domain in solution; SAXS modeling shows dimeric ALIX adopts an elongated crescent shape; ALIX dimerization mutations prevent this and affect HIV-1 budding; dimeric ALIX bridges CHMP4B filaments formed in vitro, acting as a scaffold during membrane remodeling.\",\n      \"method\": \"Small-angle X-ray scattering (SAXS); dimerization mutagenesis; in vitro filament bridging assay; HIV budding assay\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — SAXS structural modeling plus in vitro reconstitution plus functional validation\",\n      \"pmids\": [\"19523902\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"ALG-2 bridges Alix and TSG101 as a Ca2+-dependent adaptor; the ALG-2-binding site on Alix (not the PSAP motif) is required for Ca2+-dependent pulldown of TSG101; dimeric ALG-2 is required for this bridging function.\",\n      \"method\": \"Strep-pulldown assays; ALG-2 knockdown; exogenous ALG-2 rescue; mutant analysis\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pull-down with knockdown and rescue, single lab, multiple methods\",\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 causing auto-inhibition of V domain binding to viral late domains; mutations destabilizing this closed conformation open the V domain, increase ALIX membrane association, and enhance virus budding.\",\n      \"method\": \"Mutagenesis; in vitro binding assays; virus budding assays; membrane association assay\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis with functional budding assay and membrane fractionation, single lab\",\n      \"pmids\": [\"21715492\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"ALIX contains an intramolecular interaction between Patch 2 of the Bro1 domain and the TSG101-docking site in the PRD that locks ALIX in a closed conformation, preventing CHMP4 and retroviral Gag protein binding; relieving this autoinhibition promotes ALIX interaction with partners and membrane association, and increases EIAV budding.\",\n      \"method\": \"ALIX truncation/mutation analysis; co-IP; membrane fractionation; EIAV budding assay\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis with co-IP and functional assay, single lab\",\n      \"pmids\": [\"20929444\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Nedd4-1 binds ALIX directly (co-IP and yeast two-hybrid); Nedd4-1 is required for ALIX ubiquitination; ALIX recruits Nedd4-1 through the LYPX(n)L motif to facilitate HIV-1 release; Nedd4-1 catalytic activity is required for this function.\",\n      \"method\": \"Co-immunoprecipitation; yeast two-hybrid; siRNA knockdown; HIV budding assay; catalytic mutant\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP plus siRNA plus functional assay, single lab\",\n      \"pmids\": [\"20519395\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ALIX binds a YPX(3)L motif in the GPCR PAR1 via its central V domain to mediate ubiquitin-independent, ESCRT-III-dependent MVB/lysosomal sorting of PAR1; depletion of ALIX impairs PAR1 sorting into ILVs and lysosomal degradation.\",\n      \"method\": \"siRNA depletion; site-directed mutagenesis of YPX(3)L; co-immunoprecipitation; MVB sorting assay\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA plus mutagenesis plus sorting assay, single lab\",\n      \"pmids\": [\"22547407\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The V domain of ALIX binds directly and selectively to K63-linked polyubiquitin chains (preference for chains >3 Ub); mutations in two putative Ub-binding sites on a helical surface of the V domain inhibit polyUb binding in vitro and impair lentiviral budding in cells.\",\n      \"method\": \"Affinity capture/mass spectrometry; in vitro binding assays; mutagenesis; lentiviral budding assay\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro binding plus mutagenesis plus functional assay, single lab\",\n      \"pmids\": [\"23201121\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Yeast Bro1 (Alix ortholog) functions as a ubiquitin receptor via the N-terminal trihelical arm of its V domain; Bro1 binds ubiquitin and sorts ubiquitinated cargo in parallel with ESCRT-0, demonstrated by structural studies, mutagenesis, and synthetic genetic interactions with ESCRT-0.\",\n      \"method\": \"Multiple structural approaches (X-ray/NMR); mutagenesis; genetic epistasis (synthetic lethality screen)\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — structure plus mutagenesis plus genetic epistasis, single comprehensive study\",\n      \"pmids\": [\"23726974\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The syndecan-syntenin-ALIX pathway controls exosome biogenesis and ILV budding; ARF6 and its effector PLD2 regulate syntenin exosome production by controlling ILV budding into MVBs in an ALIX-dependent manner; this pathway does not affect HIV-1 budding.\",\n      \"method\": \"siRNA depletion; overexpression; electron microscopy; exosome isolation\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA with multiple readouts, single lab\",\n      \"pmids\": [\"24637612\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"ATG12-ATG3 conjugate interacts with Alix (PDCD6IP) and controls multiple Alix-dependent processes including late endosome distribution, exosome biogenesis, and viral budding; Alix is functionally required for efficient basal (but not starvation-induced) autophagy.\",\n      \"method\": \"Co-immunoprecipitation; siRNA/knockout; autophagy flux assay; exosome isolation; electron microscopy\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus KO with defined phenotypic readouts, single lab\",\n      \"pmids\": [\"25686249\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Heparanase stimulates syndecan-syntenin-ALIX-dependent exosome production by trimming heparan sulfate chains on syndecans, promoting intraluminal budding of syndecan and syntenin-1 in an ALIX-dependent manner.\",\n      \"method\": \"siRNA depletion; heparanase treatment; exosome isolation and quantification; electron microscopy\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA with multiple readouts plus biochemical rescue, single lab\",\n      \"pmids\": [\"25732677\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"ALG-2 calcium-dependent interaction with Alix relieves ALIX intramolecular autoinhibition, promoting CHMP4-dependent ALIX membrane association and MVB sorting of activated EGFR; this ALG-2 activation of ALIX is specific to MVB sorting and does not affect cytokinetic abscission or EIAV budding.\",\n      \"method\": \"Co-immunoprecipitation; membrane fractionation; MVB sorting assay; siRNA; mutagenesis\",\n      \"journal\": \"Cell discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus functional sorting assay plus pathway specificity demonstration, single lab\",\n      \"pmids\": [\"27462417\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"ARRDC3 (α-arrestin) regulates ALIX ubiquitination by WWP2 E3 ligase; ARRDC3 and WWP2 are required for ALIX interaction with activated PAR1 and CHMP4B, and for PAR1 lysosomal sorting; WWP2 interacts with ARRDC3 (not ALIX) to ubiquitinate ALIX.\",\n      \"method\": \"siRNA screen; co-immunoprecipitation; ubiquitination assay; GPCR sorting assay\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA plus co-IP plus ubiquitination assay, single lab\",\n      \"pmids\": [\"26490116\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"ALIX is required for abscission completion in Drosophila female germline stem cells (fGSCs) in vivo; ALIX and ESCRT-III component Shrub interact directly, co-localize at midbody rings, and their direct interaction is required for normal abscission kinetics.\",\n      \"method\": \"Drosophila loss-of-function genetics; co-immunoprecipitation; fluorescence microscopy in vivo\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo genetic loss-of-function with co-IP and localization, single lab\",\n      \"pmids\": [\"25635693\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ALIX and ESCRT-I/II function as parallel ESCRT-III recruiters during cytokinetic abscission; ALIX specifically recruits CHMP4C (abscission checkpoint component) while ESCRT-II/CHMP6 cooperate with ESCRT-I to recruit CHMP4B; ALIX depletion causes furrow regression in cells with chromosome bridges, implicating ALIX in abscission checkpoint signaling.\",\n      \"method\": \"siRNA depletion; fluorescence microscopy; cytokinesis/abscission assay; epistasis analysis\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA epistasis with defined phenotypic readouts, single lab\",\n      \"pmids\": [\"26929449\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ALIX mediates ubiquitin-independent lysosomal sorting of the P2Y1 GPCR via a YPX3L motif in the second intracellular loop; site-directed mutation of this motif or ALIX depletion inhibits P2Y1 MVB/lysosomal sorting.\",\n      \"method\": \"siRNA depletion; site-directed mutagenesis; MVB sorting/lysosomal degradation assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA plus mutagenesis with functional readout, single lab\",\n      \"pmids\": [\"27301021\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Alix binds Ago2 (Argonaute 2) by co-immunoprecipitation; ALIX knockdown decreases miRNA content in extracellular vesicles without reducing vesicle number, indicating a role for Alix in miRNA packaging into EVs.\",\n      \"method\": \"Co-immunoprecipitation; siRNA knockdown; RT-qPCR for miRNAs in EVs\",\n      \"journal\": \"International journal of molecular medicine\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single co-IP and siRNA experiment, single lab, single method per claim\",\n      \"pmids\": [\"26935291\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Alix is required for clathrin-independent endocytosis (CIE) but not clathrin-mediated endocytosis; in Alix knockout cells, fluid-phase endocytosis and CIE cargoes (cholera toxin, IL-2 receptor) are impaired; Alix acts with endophilin-A to promote CIE and regulate cell migration.\",\n      \"method\": \"Alix knockout cells; endocytosis assays; signaling assays; cell migration assay\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with defined endocytic phenotype and multiple cargoes tested, 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 assemble the apically restricted actomyosin-tight junction complex in epithelial cells; Alix knockout mice develop hydrocephalus due to defects in epithelial polarity, tight junction formation, and excessive cell extrusion in choroid plexus/ependyma.\",\n      \"method\": \"Knockout mouse model; co-immunoprecipitation; fluorescence/electron microscopy; phenotypic analysis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse with co-IP and defined structural phenotype, single lab\",\n      \"pmids\": [\"27336173\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Alix undergoes S-palmitoylation as a post-translational modification; this lipid modification influences Alix protein-protein interaction with CD9 (tetraspanin) and affects the structural organization of the lipid bilayer of extracellular vesicles.\",\n      \"method\": \"Palmitoylation assay (acyl-RAC); co-immunoprecipitation; biophysical lipid bilayer analysis\",\n      \"journal\": \"Biochimica et biophysica acta. General subjects\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — palmitoylation assay plus co-IP, single lab, limited mechanistic depth\",\n      \"pmids\": [\"30251702\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"ALIX depletion results in prolonged EGFR activity after stimulation, defective PD-L1 trafficking through MVBs, reduced PD-L1 exosomal secretion, and redistribution of PD-L1 to the cell surface, conferring an EGFR-dependent immunosuppressive phenotype.\",\n      \"method\": \"siRNA depletion; EGFR signaling assays; flow cytometry for PD-L1; exosome isolation; mouse tumor model\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA with multiple readouts including in vivo model, single lab\",\n      \"pmids\": [\"30021161\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"In Drosophila, ALIX is recruited to the midbody via its V domain through direct interaction with an LxxLF motif in centralspindlin component Pavarotti (MKLP1), using the same conserved hydrophobic V-domain pocket that binds LYPXnL virus proteins; this mechanism is CEP55-independent.\",\n      \"method\": \"Co-immunoprecipitation; domain mapping/mutagenesis; fluorescence microscopy; Drosophila genetics\",\n      \"journal\": \"Current biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP with domain mutagenesis and in vivo localization, single lab\",\n      \"pmids\": [\"31607533\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Phosphorylated MLKL (necroptosis mediator) is removed from membranes via ALIX-syntenin-1-mediated exocytosis; ALIX and syntenin-1 are detected in membrane-localized MLKL immunoprecipitates by protein cross-linking/affinity purification.\",\n      \"method\": \"Protein cross-linking; affinity purification; functional exocytosis assay\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — crosslinking-AP plus functional assay, single lab\",\n      \"pmids\": [\"31138766\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ALIX (active form) recruits ESCRT-III proteins to endosomes independently of other ESCRTs but requiring lysobisphosphatidic acid (LBPA) in vivo; this can be reconstituted on supported bilayers in vitro; this ALIX-ESCRT-III pathway promotes sorting of tetraspanins specifically to exosomes.\",\n      \"method\": \"In vitro reconstitution on supported bilayers; ALIX activation mutants; siRNA; electron microscopy\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution on bilayers plus in vivo siRNA with cargo-specific readout, single comprehensive study\",\n      \"pmids\": [\"32049272\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The ESCRT-III machinery localizes to the abscission site via a plasma membrane-to-ESCRT module composed of syndecan-4 (transmembrane proteoglycan), ALIX, and syntenin; direct interactions between ALIX, syntenin, and syndecan-4 are essential for ESCRT-III enrichment at the abscission site (but not at the midbody); depletion of any of the three proteins delays abscission.\",\n      \"method\": \"Proteomics of isolated midbodies (Flemmingsome); siRNA depletion; co-immunoprecipitation; fluorescence microscopy\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus siRNA with defined abscission phenotype plus proteomic identification, single lab\",\n      \"pmids\": [\"32321914\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ALIX and Syntenin-1 are required for proper subcellular localization and efficient EV secretion of APP; their knockdown sequesters APP in ER and endolysosomal compartments respectively; this pathway is ESCRT-independent.\",\n      \"method\": \"Inducible siRNA knockdown; subcellular localization; EV isolation and analysis\",\n      \"journal\": \"BMC molecular and cell biology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — siRNA with localization readout, single lab, indirect mechanistic evidence\",\n      \"pmids\": [\"32731849\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The ALIX proline-rich domain (PRD) contains three tandem proline-rich motifs that compete for a single binding site on TSG101-UEV; the C-terminal tyrosine-rich portion of ALIX-PRD forms amyloid fibrils that dissolve upon hyperphosphorylation by Src kinase, revealing a phosphorylation-controlled reversible amyloid mechanism regulating ALIX function.\",\n      \"method\": \"Heteronuclear NMR spectroscopy; relaxation dispersion; thioflavin T/Congo red assays; electron microscopy; Src kinase phosphorylation\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — NMR with kinetic measurements, in vitro reconstitution of amyloid formation/dissolution, mutagenesis, multiple orthogonal methods\",\n      \"pmids\": [\"32917811\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ALIX (not the broader ESCRT machinery) is required specifically for apical exosome release in polarized epithelial cells via the ALIX-Syntenin1-Syndecan1 machinery, while basolateral exosome release depends on ceramide; these represent two independent polarized exosome release pathways.\",\n      \"method\": \"siRNA depletion; polarized epithelial cell model; exosome isolation; directional secretion assay\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA with directional exosome assay in polarized cells, single lab\",\n      \"pmids\": [\"33724661\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Alix is a substrate of the muscle-specific ubiquitin E3 ligase Ozz-E3; Ozz interaction with Alix changes Alix conformation and promotes its ubiquitination, regulating Alix levels at cortactin-containing compartments; Alix knockdown in muscle cells affects F-actin amount and distribution, impairs sarcolemmal protrusion formation and cell motility.\",\n      \"method\": \"Co-immunoprecipitation (endogenous proteins); ubiquitination assay; Ozz knockout mouse; siRNA knockdown; actin/morphology assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — endogenous co-IP plus KO mouse plus siRNA with functional readout, single lab\",\n      \"pmids\": [\"22334701\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"SUMOylated hnRNPA2B1 (SUMO2 at K108) is recognized by the SUMO interaction motif (SIM) of ALIX, activating ALIX to recruit ESCRT-III for sorting of circTLCD4-RWDD3 into extracellular vesicles; mutating the SIM in ALIX or K108 in hnRNPA2B1 blocks EV packaging and inhibits lymphatic metastasis in vivo.\",\n      \"method\": \"Co-immunoprecipitation; SUMOylation assay; SIM mutation in ALIX; in vivo tumor model\",\n      \"journal\": \"Signal transduction and targeted therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP with mutagenesis plus in vivo validation, single lab\",\n      \"pmids\": [\"37925421\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PDCD6IP/Alix is a multifunctional ESCRT-associated adaptor protein that operates as a conformationally regulated scaffold: its N-terminal Bro1 domain binds CHMP4/ESCRT-III subunits (using a hydrophobic code distinct from MIT-domain partners), its central V domain binds YPX(n)L late-domain motifs of retroviruses and cellular receptors (including GPCRs via YPX(n)L sequences) as well as K63-linked polyubiquitin chains, and its C-terminal proline-rich domain (PRD) engages ALG-2 (in a Ca2+-dependent arginine-switch mechanism), TSG101, endophilins, and CEP55/centralspindlin; the PRD also folds back onto the Bro1/V domains to maintain an autoinhibited closed conformation that is relieved by Ca2+/ALG-2, membrane recruitment, partner binding, or Ozz/Nedd4/WWP2-mediated ubiquitination, thereby activating ALIX to recruit ESCRT-III and drive membrane fission during enveloped virus budding, MVB intraluminal vesicle formation (including a tetraspanin-specific pathway requiring LBPA), exosome biogenesis (via syndecan-syntenin-ALIX and ARF6-PLD2 axes), cytokinetic abscission (recruited by CEP55 in mammals or centralspindlin in Drosophila to the midbody), clathrin-independent endocytosis, and actin cytoskeleton organization.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PDCD6IP/Alix is a conformationally regulated ESCRT-associated scaffold that links membrane cargo and accessory proteins to the ESCRT-III machinery to drive membrane fission across multiple cellular processes [#12, #43]. Its architecture comprises an N-terminal Bro1 domain that binds CHMP4/ESCRT-III subunits through a hydrophobic code distinct from the MIT-domain partners of VPS4 [#16], a central V domain that recognizes YPX(n)L late-domain motifs of retroviral Gag proteins and cellular GPCRs as well as K63-linked polyubiquitin chains [#12, #14, #25, #26], and a C-terminal proline-rich domain that engages ALG-2 in a Ca2+-dependent arginine-switch manner, TSG101, and endophilins [#1, #6, #17, #46]. The proline-rich region folds back onto the Bro1/V domains to lock Alix in an autoinhibited closed conformation that is relieved by Ca2+/ALG-2, membrane recruitment, partner binding, or E3-ligase-mediated ubiquitination, thereby licensing CHMP4 recruitment and membrane association [#22, #23, #31, #48]. Through these interactions Alix mediates enveloped virus budding [#2, #12], multivesicular body intraluminal vesicle formation and a tetraspanin-specific exosome pathway requiring lysobisphosphatidic acid [#18, #43], syndecan-syntenin-driven exosome biogenesis [#28, #30], ubiquitin-independent MVB/lysosomal sorting of GPCRs via YPX(n)L motifs [#25, #35], and cytokinetic abscission, where it is recruited to the midbody by CEP55 in mammals or centralspindlin in Drosophila and delivers CHMP4 to the abscission site [#13, #34, #41]. Alix additionally organizes the actin cytoskeleton and epithelial junctions; its loss in mice produces hydrocephalus from defects in epithelial polarity and tight-junction assembly [#11, #38], and it supports clathrin-independent endocytosis together with endophilin-A [#37].\"\n,\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Established Alix's founding biochemical partnership, defining it as a Ca2+-regulated effector and hinting at a role in regulated death/signaling.\",\n      \"evidence\": \"Yeast two-hybrid and co-IP identifying ALG-2 as a calcium-dependent partner\",\n      \"pmids\": [\"10200558\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of the ALG-2 interaction unresolved at this stage\", \"No structural basis for Ca2+ dependence yet\", \"No link to membrane trafficking established\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Mapped the proline-rich domain as a protein-interaction hub by identifying endophilin binding, linking Alix to membrane-deforming machinery.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP and overlay assays with PXRPPPP consensus mapping\",\n      \"pmids\": [\"12034747\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological process served by Alix-endophilin binding undefined\", \"No structural detail of the interface\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Placed Alix in the ESCRT pathway by showing it bridges retroviral Gag late domains to ESCRT-III and TSG101, defining its role in viral budding.\",\n      \"evidence\": \"Co-IP, binding and functional budding assays with HIV-1 p6, EIAV p9, and CHMP4b\",\n      \"pmids\": [\"14505569\", \"12860994\", \"12588984\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic basis of YPX(n)L and CHMP4 recognition not yet known\", \"How budding is spatially coordinated unresolved\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Defined the ALG-2 binding site on Alix and uncovered counter-roles in receptor downregulation, endosome organization, and non-apoptotic death, broadening Alix beyond budding.\",\n      \"evidence\": \"Mutagenesis/overlay/co-IP mapping ALG-2 to residues 794-827; siRNA and ubiquitination assays for EGFR/Cbl; Src kinase assays; paraptosis assays\",\n      \"pmids\": [\"14999017\", \"15456872\", \"15557335\", \"15195070\", \"15086793\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanistic link between EGFR regulation and ESCRT sorting unclear\", \"Src phosphorylation site and structural effect not resolved at this stage\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Connected Alix to endosome positioning and the lipid environment of MVBs, distinguishing its role from EGFR degradation and recycling.\",\n      \"evidence\": \"RNAi with immunofluorescence and immunoelectron microscopy measuring LBPA and endosome distribution\",\n      \"pmids\": [\"15914539\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis of LBPA dependence undefined\", \"Mechanism linking Alix loss to aberrant actin/clathrin structures unclear\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Demonstrated direct actin-cytoskeleton engagement, establishing a structural role for Alix beyond membrane scission.\",\n      \"evidence\": \"Cell-free direct binding to F-actin, cortactin, alpha-actinin; siRNA phenotypes on stress fibers\",\n      \"pmids\": [\"16966331\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How cytoskeletal and ESCRT functions are coordinated unknown\", \"In vivo relevance not addressed here\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Solved the structural logic of Alix: defined Bro1 and V domain architecture, the YPX(n)L pocket, CHMP4 binding, and the CEP55-dependent midbody recruitment for abscission.\",\n      \"evidence\": \"Crystallography of Bro1/V domains and V-domain:late-domain complexes; proteomics, co-IP, siRNA and cytokinesis assays for CEP55/TSG101; NC-Bro1 packaging\",\n      \"pmids\": [\"17350572\", \"17277784\", \"18066081\", \"17853893\", \"18032513\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Conformational regulation of these interactions not yet defined\", \"Cargo-specific pathway selection unresolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Defined the molecular grammar of partner selection and oligomeric scaffolding: the CHMP4 hydrophobic code, the ALG-2 arginine switch, V-domain dimerization, and a positive/negative regulatory split with TSG101 in ILV budding.\",\n      \"evidence\": \"Crystallography of Bro1:CHMP4 and Ca2+/ALG-2 states; SAXS dimer modeling and filament bridging; in vitro ILV reconstitution; abscission/HIV mutagenesis\",\n      \"pmids\": [\"18511562\", \"18940611\", \"18768755\", \"18641129\", \"19523902\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How dimerization is triggered in cells unclear\", \"Reconciliation of Alix as negative regulator in vitro vs positive elsewhere\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Identified intramolecular autoinhibition as the master regulatory principle, with the PRD folding back to occlude V and Bro1 surfaces and ubiquitination/E3 ligases as activating inputs.\",\n      \"evidence\": \"Truncation/mutagenesis with co-IP, membrane fractionation, budding assays; Nedd4-1 and ALG-2 bridging studies\",\n      \"pmids\": [\"21715492\", \"20929444\", \"20519395\", \"19520058\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Quantitative dynamics of open/closed equilibrium not measured\", \"Cell-type-specific activation inputs incompletely mapped\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Extended Alix cargo recognition to ubiquitin-independent GPCR sorting via YPX(3)L motifs and to direct K63-polyubiquitin binding, and identified Ozz-E3 ubiquitination as a conformational/abundance regulator.\",\n      \"evidence\": \"siRNA/mutagenesis PAR1 sorting; in vitro polyUb binding and mutagenesis; endogenous co-IP, Ozz KO mouse and actin phenotypes\",\n      \"pmids\": [\"22547407\", \"23201121\", \"22334701\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relative contributions of ubiquitin-dependent vs -independent cargo capture unclear\", \"Tissue-specific roles of Ozz regulation undefined\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Established the ancestral V-domain ubiquitin-receptor function via the yeast Bro1 ortholog, paralleling ESCRT-0 in cargo sorting.\",\n      \"evidence\": \"Structural studies, mutagenesis, and synthetic genetic epistasis with ESCRT-0 in yeast\",\n      \"pmids\": [\"23726974\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Conservation of this ubiquitin-receptor role in human Alix not formally proven here\", \"Interplay with V-domain YPX(n)L binding unresolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Defined the syndecan-syntenin-ALIX exosome pathway and its ARF6-PLD2 control, establishing a budding route distinct from viral budding.\",\n      \"evidence\": \"siRNA, overexpression, EM and exosome isolation\",\n      \"pmids\": [\"24637612\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How ARF6-PLD2 lipids feed into Alix activation unclear\", \"Cargo selectivity of this pathway incompletely defined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Integrated Alix into autophagy, basal endosomal exosome biogenesis, and demonstrated pathway-selective activation, while resolving exosome production regulators including heparanase and parallel abscission recruiters.\",\n      \"evidence\": \"ATG12-ATG3 co-IP/KO; ALG-2 activation specific to EGFR MVB sorting; heparanase exosome assays; Drosophila Shrub genetics; CHMP4C-specific abscission siRNA epistasis; ARRDC3/WWP2 ubiquitination\",\n      \"pmids\": [\"25686249\", \"27462417\", \"25732677\", \"25635693\", \"26929449\", \"26490116\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How a single scaffold achieves pathway-specific activation mechanistically unclear\", \"Crosstalk between activating inputs (ALG-2, ubiquitination, lipids) unresolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Broadened Alix's cellular roles to clathrin-independent endocytosis, epithelial junction/actin assembly with an in vivo hydrocephalus phenotype, additional GPCR sorting, and EV miRNA packaging.\",\n      \"evidence\": \"Alix KO endocytosis assays with endophilin-A; KO mouse epithelial polarity/tight-junction analysis; P2Y1 YPX3L sorting; Ago2 co-IP and EV miRNA quantification\",\n      \"pmids\": [\"27244115\", \"27336173\", \"27301021\", \"26935291\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanistic link between Alix's actin-junction role and its ESCRT scaffold function unclear\", \"Ago2/miRNA-packaging mechanism rests on a single low-confidence study\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Linked Alix-dependent MVB sorting to immune regulation through PD-L1 trafficking and EV secretion, and identified S-palmitoylation as a lipid-modification input.\",\n      \"evidence\": \"siRNA, flow cytometry, exosome isolation and mouse tumor model for PD-L1; acyl-RAC palmitoylation and CD9 co-IP\",\n      \"pmids\": [\"30021161\", \"30251702\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Palmitoylation site and functional requirement rest on a single low-confidence study\", \"How Alix selects PD-L1 cargo unresolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Revealed an evolutionarily flexible recruitment logic — the V-domain LYPX(n)L pocket also captures the centralspindlin LxxLF motif for CEP55-independent midbody recruitment — and removal of phospho-MLKL via Alix-syntenin exocytosis.\",\n      \"evidence\": \"Co-IP, domain mutagenesis and Drosophila genetics for Pavarotti; cross-linking/AP and exocytosis assay for MLKL\",\n      \"pmids\": [\"31607533\", \"31138766\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether mammalian centralspindlin uses the same pocket unresolved\", \"Regulation switching between viral and midbody ligands at the same pocket unclear\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Reconstituted active Alix recruiting ESCRT-III on LBPA-containing bilayers for tetraspanin exosome sorting, defined the syndecan-4-syntenin-Alix module at the abscission site, and resolved a phosphorylation-controlled reversible amyloid in the PRD that regulates TSG101 competition.\",\n      \"evidence\": \"In vitro bilayer reconstitution and siRNA/EM for tetraspanins; Flemmingsome proteomics and abscission siRNA; NMR/relaxation/ThT and Src phosphorylation of the PRD\",\n      \"pmids\": [\"32049272\", \"32321914\", \"32917811\", \"32731849\", \"32049272\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological trigger of PRD amyloid formation in cells unclear\", \"How the syndecan-4 module is distinguished from midbody recruitment incompletely defined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Showed Alix governs a polarity-specific exosome route, mediating apical exosome release distinct from ceramide-dependent basolateral release.\",\n      \"evidence\": \"siRNA and directional exosome assays in polarized epithelial cells\",\n      \"pmids\": [\"33724661\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis of apical vs basolateral sorting unresolved\", \"Link to the epithelial junction role of Alix not connected\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identified a SUMO-dependent cargo-capture mode in which the Alix SIM recognizes SUMOylated hnRNPA2B1 to drive circRNA packaging into EVs with pro-metastatic consequences.\",\n      \"evidence\": \"Co-IP, SUMOylation assays, SIM mutation and in vivo metastasis model\",\n      \"pmids\": [\"37925421\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Generality of the SIM-dependent cargo route beyond this circRNA unknown\", \"How SUMO recognition integrates with autoinhibition release unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a single autoinhibited scaffold integrates Ca2+/ALG-2, ubiquitination, SUMO, lipid, and phosphorylation/amyloid inputs to select among virus budding, ILV/exosome biogenesis, abscission, endocytosis, and cytoskeletal roles in a given cell remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified quantitative model of conformational switching across pathways\", \"Cargo- and pathway-specific activation inputs only partly mapped\", \"Mammalian counterpart of several Drosophila/yeast findings not formally confirmed\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [2, 12, 13, 17, 21]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [11, 38, 48]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [20, 43]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [7, 18]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [3, 10, 25, 31]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [22, 38, 44]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [9, 10]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [11, 48]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [3, 18, 28, 43]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [13, 34, 44]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [2, 12, 14]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [7, 25, 35]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [36, 49]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [29]}\n    ],\n    \"complexes\": [\"ESCRT-III (CHMP4)\", \"syndecan-syntenin-ALIX module\", \"actomyosin-tight junction complex\"],\n    \"partners\": [\"PDCD6 (ALG-2)\", \"CHMP4B\", \"TSG101\", \"CEP55\", \"SDCBP (syntenin)\", \"endophilin\", \"NEDD4\", \"PAR1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}