{"gene":"PDCD6","run_date":"2026-06-10T05:19:53","timeline":{"discoveries":[{"year":1996,"finding":"ALG-2 (PDCD6) encodes a Ca2+-binding protein required for T cell receptor-, Fas-, and glucocorticoid-induced apoptosis; ALG-2-depleted 3DO T cell hybridoma cells are protected from these death stimuli, placing ALG-2 as a required mediator of Ca2+-regulated apoptotic signaling.","method":"Functional selection (death trap assay), antisense depletion of ALG-2 with loss-of-function apoptosis phenotype","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 / Strong — foundational loss-of-function study with clear apoptosis phenotype, replicated across multiple death stimuli, widely cited and independently confirmed","pmids":["8560270"],"is_preprint":false},{"year":1997,"finding":"ALG-2 functions downstream of or independently of ICE/Ced-3 (caspase) activation during apoptosis; ALG-2-depleted cells exhibit normal caspase-mediated PARP cleavage yet are protected from cell death, indicating ALG-2 acts at a post-caspase step.","method":"Caspase activity assay (fluorogenic substrate, PARP cleavage) in ALG-2-depleted T cell hybridoma clones","journal":"Journal of Immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct biochemical epistasis experiment using endogenous substrate cleavage assays, consistent with and extending the founding paper","pmids":["9164928"],"is_preprint":false},{"year":1998,"finding":"Ca2+ binding to ALG-2 induces exposure of a hydrophobic surface on the protein, as detected by a fluorescent hydrophobicity probe (TNS); this conformational change occurs with half-maximal effect at ~6 µM Ca2+, and Mg2+ is not effective.","method":"Fluorescence spectroscopy with hydrophobicity probe TNS; gel filtration; biochemical fractionation of mammalian cells","journal":"Journal of Biochemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro biophysical characterization, single lab, but clear mechanistic result on Ca2+-dependent conformational change","pmids":["9832622"],"is_preprint":false},{"year":1999,"finding":"ALG-2 interacts with AIP1/Alix in a strictly Ca2+-dependent manner; both proteins co-localize in the cytosol; overexpression of a truncated AIP1 protects cells from trophic factor withdrawal-induced death, indicating AIP1 cooperates with ALG-2 in the Ca2+-dependent cell death pathway.","method":"Yeast two-hybrid screening, co-immunoprecipitation, subcellular co-localization, overexpression rescue assay","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP plus functional rescue, independently confirmed by parallel study (PMID:10200558), multiple methods","pmids":["9880530","10200558"],"is_preprint":false},{"year":1999,"finding":"ALG-2 possesses two high-affinity Ca2+-binding sites; Ca2+ binding induces conformational changes in both N- and C-terminal domains; the fifth EF-hand participates in homodimerization; Ca2+ binding to both strong sites is required for Ca2+-induced protein aggregation.","method":"Recombinant protein expression, gel filtration, chemical cross-linking, fluorescence spectroscopy, circular dichroism, site-directed mutagenesis (Glu47Ala/Glu114Ala)","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with mutagenesis and multiple spectroscopic methods, single lab","pmids":["10360947"],"is_preprint":false},{"year":2000,"finding":"Two alternatively spliced isoforms of ALG-2 exist (ALG-2,5 full-length and ALG-2,1 lacking Gly121/Phe122); they differ in Ca2+ affinity; ALG-2,1 does not interact with AIP1/Alix despite otherwise similar properties, indicating these two residues are critical for target recognition.","method":"cDNA cloning, Ca2+ binding assays, yeast two-hybrid interaction assay","journal":"The Journal of Biological Chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid plus Ca2+ binding biochemistry, single lab, two orthogonal methods","pmids":["10744743"],"is_preprint":false},{"year":2001,"finding":"X-ray crystal structure of Ca2+-loaded ALG-2 (des1-20, 2.3 Å) reveals five EF-hands folding into eight α-helices, dimer formation, Ca2+ binding to EF1, EF3, and EF5; a rigid-body rotation of EF1-2 relative to EF4-5 (hinge at EF3) upon Ca2+ loading exposes a hydrophobic patch and a large cleft near the dimer interface that accommodates a Gly/Pro-rich peptide.","method":"X-ray crystallography at 2.3 Å resolution; limited proteolysis for crystal preparation","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with mechanistic interpretation of Ca2+-dependent conformational change, foundational structural paper","pmids":["11525164"],"is_preprint":false},{"year":2001,"finding":"ALG-2 forms a Ca2+-independent homodimer and a Ca2+-dependent heterodimer with its closest paralog peflin; peflin dissociates from ALG-2 in the presence of Ca2+; peflin translocates to membrane/cytoskeletal fractions in Ca2+ conditions while ALG-2 is also found in the nucleus.","method":"Co-immunoprecipitation with monoclonal anti-peflin antibody, epitope-tag co-IP, gel filtration, immunofluorescence, subcellular fractionation","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP plus gel filtration plus fractionation, multiple orthogonal methods, single lab","pmids":["11278427"],"is_preprint":false},{"year":2002,"finding":"ALG-2 interacts directly and Ca2+-dependently with the N-terminal domain of annexin XI; KD ~70 nM for the high-affinity site; the Pro/Gly/Tyr/Ala-rich hydrophobic region of annexin XI masks Ca2+-dependently exposed hydrophobic surface of ALG-2.","method":"Yeast two-hybrid, GST pull-down, biotin-ALG-2 overlay assay, surface plasmon resonance (SPR), fluorescence probe inhibition","journal":"Biochemical and Biophysical Research Communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct binding reconstitution with SPR kinetics plus overlay, single lab but multiple orthogonal methods","pmids":["11883939"],"is_preprint":false},{"year":2002,"finding":"ALG-2 binds directly and Ca2+-dependently to the N-terminal domains of both annexin VII and annexin XI with two binding sites (KD ~40-60 nM high-affinity, ~500-700 nM low-affinity), establishing a common PGAYQ-biased binding mode.","method":"Biotin-ALG-2 overlay assay, surface plasmon resonance biosensor with GST-fusion proteins","journal":"Biochimica et Biophysica Acta","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with SPR kinetics and overlay, single lab, two orthogonal methods","pmids":["12445460"],"is_preprint":false},{"year":2002,"finding":"The fifth EF-hand (EF-5) of both ALG-2 and peflin is essential for dimerization and protein stability; EF-5 deletion mutants are rapidly degraded by the proteasome.","method":"Deletion mutagenesis, pulse-chase experiment, proteasome inhibitor (MG132) treatment, Western blot, subcellular fractionation","journal":"Archives of Biochemistry and Biophysics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function mutagenesis with pharmacological rescue, single lab","pmids":["11883899"],"is_preprint":false},{"year":2002,"finding":"ALG-2 interacts with ASK1 (apoptosis signal-regulating kinase 1) at its C-terminus (aa 941-1375) in a Ca2+-dependent manner; the ALG-2,1 isoform lacking Gly121/Phe122 does not bind ASK1; co-expression of ALG-2 redirects ASK1 to the nucleus and inhibits ASK1-induced JNK activation.","method":"Co-immunoprecipitation in BOSC23 cells, in vitro binding, co-transfection with JNK activity readout, subcellular localization by microscopy","journal":"FEBS Letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus functional JNK assay, isoform specificity demonstrated, single lab","pmids":["12372597"],"is_preprint":false},{"year":2004,"finding":"ALG-2 interacts directly with Alix through a region (aa 794-827) containing four tandem PxY repeats; seven proline and four tyrosine residues in PxY repeats are critical for binding affinity; Ca2+-binding-deficient ALG-2(E47A/E114A) does not co-immunoprecipitate with Alix; ALG-2 is required for the subcellular punctate distribution of the Alix C-terminal half.","method":"Yeast two-hybrid, biotin-ALG-2 overlay, alanine-substitution mutagenesis, co-immunoprecipitation, GFP-fusion fluorescence microscopy","journal":"Journal of Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct binding reconstitution with mutagenesis plus Co-IP plus imaging, multiple orthogonal methods in one study","pmids":["14999017"],"is_preprint":false},{"year":2005,"finding":"ALG-2 directly binds the proline-rich region (PRR) of TSG101 (ESCRT-I component) in a Ca2+-dependent manner; ALG-2 also associates with hVps28 and hVps37A indirectly via TSG101; ALG-2 co-localizes with SKD1(E235Q)-induced aberrant endosomes in a Ca2+-dependent manner; Ca2+ chelation abolishes this punctate endosomal localization.","method":"GST pull-down, yeast two-hybrid, biotin-ALG-2 overlay assay, immunofluorescence microscopy, Ca2+ chelator (BAPTA-AM) treatment","journal":"The Biochemical Journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct binding demonstrated by overlay plus pulldown plus yeast two-hybrid, subcellular localization with Ca2+ dependence, multiple methods","pmids":["16004603"],"is_preprint":false},{"year":2006,"finding":"ALG-2 is recruited to ER exit sites (ERES) via Ca2+-dependent interaction with Sec31A (COPII outer coat component); ALG-2 in turn stabilizes Sec31A at ERES; Ca2+-binding-deficient ALG-2 mutant loses ERES localization; Ca2+ chelation disperses ALG-2 and reduces membrane-associated Sec31A.","method":"Ca2+-dependent GST pull-down, biotin-ALG-2 overlay, immunofluorescence confocal microscopy, RNAi knockdown, Ca2+ ionophore/chelator treatments, overexpression of Sec31A PRR as dominant negative","journal":"Molecular Biology of the Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (pulldown, overlay, RNAi, imaging, pharmacological), replicated by independent concurrent study (PMID:17196169)","pmids":["16957052","17196169"],"is_preprint":false},{"year":2006,"finding":"ALG-2 subcellular distribution oscillates between cytosol and punctate (COPII/Sec31A-positive) localization in phase with intracellular Ca2+ oscillations triggered by physiological stimuli (ATP, EGF, prostaglandin, histamine); a Ca2+-binding-deficient mutant does not redistribute.","method":"Live-cell fluorescence imaging of tagged ALG-2 simultaneous with Ca2+ indicators; Ca2+-binding-deficient mutant as control","journal":"Biochemical and Biophysical Research Communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live-cell imaging with Ca2+ co-monitoring and mutant control, single lab","pmids":["17214967"],"is_preprint":false},{"year":2006,"finding":"ALG-2 is translocated to the nucleus upon co-expression with the RNA-binding protein RBM22, which shuttles between cytoplasm and nucleus; in zebrafish embryos the two proteins co-localize within the nucleus, suggesting RBM22-mediated nuclear import of ALG-2.","method":"Yeast two-hybrid screening, confocal microscopy of fluorescent fusions in NIH 3T3 cells and zebrafish embryos","journal":"Biochimica et Biophysica Acta","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-localization imaging plus yeast two-hybrid, two model systems, but no direct binding reconstitution or functional consequence established","pmids":["17045351"],"is_preprint":false},{"year":2006,"finding":"POSH scaffold protein forms a Ca2+-dependent complex with ALG-2 and ALIX in Drosophila; overexpression of ALG-2 causes roughened/melanized eye phenotypes; co-overexpression with POSH enhances these phenotypes; ALG-2 overexpression induces ectopic JNK activation, suggesting POSH/ALG-2/ALIX function together in JNK pathway regulation.","method":"Co-immunoprecipitation in Drosophila cells, genetic overexpression in eye imaginal discs, JNK activity assay","journal":"FEBS Letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus in vivo genetic epistasis in Drosophila, single lab","pmids":["16698022"],"is_preprint":false},{"year":2007,"finding":"ALG-2 binds to Scotin (a p53-inducible ER-membrane protein) in a strictly Ca2+-dependent manner; overexpression of ALG-2 increases Scotin protein levels, indicating ALG-2 stabilizes Scotin.","method":"In vitro binding assay (synthesized C-terminal Scotin fragment on immobilized ALG-2), co-immunoprecipitation in MCF7 and U2OS cell lines, overexpression Western blot","journal":"Archives of Biochemistry and Biophysics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus in vitro direct binding, multiple cell lines, Ca2+ dependence confirmed, single lab","pmids":["17889823"],"is_preprint":false},{"year":2008,"finding":"X-ray crystal structures of Ca2+-free and Ca2+-bound ALG-2 and of the ALG-2/Alix799-814 peptide complex reveal a Ca2+/EF3-driven arginine switch: Ca2+ binding to EF3 repositions Arg125, opening a primary hydrophobic pocket (Pocket 1) that accepts the PPYP motif of Alix; in vitro binding assays with mutant proteins validate this mechanism.","method":"X-ray crystallography (multiple structures), in vitro binding assay with ALG-2 and Alix mutants","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple crystal structures plus mutagenesis-validated binding mechanism, mechanistically definitive","pmids":["18940611"],"is_preprint":false},{"year":2008,"finding":"ALG-2 interacts with Alix and pro-caspase-8; Alix forms a complex with TNF-R1-containing endosomes in a manner dependent on ESCRT binding; deletion of the ALG-2-binding site on Alix significantly reduces TNF-R1-induced cell death without affecting receptor endocytosis, placing ALG-2 in the TNF-R1 death signaling pathway.","method":"Mass spectrometry of Alix co-immunoprecipitates, Co-IP, overexpression of Alix deletion mutants, cell death assays, motoneuron primary culture","journal":"The Journal of Biological Chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus domain deletion functional assay, single lab","pmids":["18936101"],"is_preprint":false},{"year":2008,"finding":"ALG-2 interacts with PLSCR3 Ca2+-dependently via two distinct binding sites: ABS-1 (PPYP-type, type 1 motif, Alix-like) recognized only by full-length ALG-2, and ABS-2 (PXPGF-type, type 2 motif) recognized by both full-length ALG-2 and the ALG-2(ΔGF122) isoform; Phe49 in ABS-2 is critical for binding.","method":"Co-immunoprecipitation, GST pull-down, biotin-ALG-2 overlay, surface plasmon resonance with synthetic oligopeptides, mutagenesis","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — SPR kinetics plus mutagenesis plus multiple pulldown methods defining two distinct binding pockets, single lab","pmids":["18256029"],"is_preprint":false},{"year":2009,"finding":"ALG-2 functions as a Ca2+-dependent adaptor protein that bridges Alix and TSG101: ALG-2 knockdown abolishes Alix-TSG101 association in pulldown assays; recombinant ALG-2 restores the interaction; the shorter ALG-2(ΔGF122) isoform and a dimerization-defective mutant cannot bridge the two proteins, indicating the ALG-2 dimer is required.","method":"Strep-tag pulldown assay with ALG-2 knockdown cells, add-back of purified recombinant ALG-2, isoform and dimerization mutant controls","journal":"Biochemical and Biophysical Research Communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — reconstitution of ternary complex with knockdown plus add-back, isoform controls, dimerization requirement demonstrated","pmids":["19520058"],"is_preprint":false},{"year":2009,"finding":"ALG-2 binds directly to the NH2-terminal cytosolic tail of mucolipin-1 (MCOLN1) Ca2+-dependently; the interaction is mediated by residues 37-49 of MCOLN1; mutation of the ALG-2-binding domain in MCOLN1 reduces MCOLN1-induced accumulation of aberrant endosomes, indicating ALG-2 modulates MCOLN1 function in the late endosomal-lysosomal pathway.","method":"Co-immunoprecipitation, direct binding assay, deletion/point mutagenesis of MCOLN1, fluorescence microscopy with dominant-negative Vps4B","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct binding plus mutagenesis with functional endosomal phenotype, multiple methods, single lab","pmids":["19864416"],"is_preprint":false},{"year":2010,"finding":"The ALG-2-binding site (ABS) in Sec31A (aa 839-851 in Pro-rich region) controls retention kinetics of Sec31A at ERES; FRAP analysis shows ABS deletion reduces the high-affinity/slow-turnover population of Sec31A at ERES.","method":"Stable cell lines with GFP-ALG-2 and Sec31A-RFP, live-cell imaging after Ca2+ mobilization, biotin-ALG-2 overlay to map ABS, FRAP","journal":"Bioscience, Biotechnology, and Biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live-cell FRAP plus overlay mapping, single lab, two orthogonal methods","pmids":["20834162"],"is_preprint":false},{"year":2010,"finding":"Crystal structure of ALG-2(ΔGF122) in Ca2+-bound form reveals that deletion of Gly121-Phe122 shortens α-helix 5 and repositions Arg125 to partially block Pocket 1, explaining failure to bind Alix; F122A/G substitutions (but not F122W) increase Alix-binding by expanding Pocket 2, while affecting binding to TSG101 and annexin A11 differently, demonstrating structural flexibility in target recognition.","method":"X-ray crystallography, in vitro binding assays with multiple ALG-2 point mutants and Alix/TSG101/annexin A11","journal":"BMC Structural Biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus systematic mutagenesis binding assays, mechanistically definitive for isoform selectivity","pmids":["20691033"],"is_preprint":false},{"year":2011,"finding":"PDCD6/ALG-2 directly binds VEGFR-2 and suppresses phosphorylation of PI3K/Akt/mTOR/GSK-3β/p70S6K signaling, reducing VEGF-induced endothelial proliferation, invasion, and tube formation in vitro.","method":"Co-immunoprecipitation to show VEGFR-2 binding, Western blot of downstream signaling, cell migration/invasion/tube formation assays with recombinant PDCD6","journal":"Cellular Signalling","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP plus signaling readouts, no direct binding reconstitution, single lab","pmids":["21893193"],"is_preprint":false},{"year":2013,"finding":"ALG-2/Ca2+ attenuates COPII vesicle budding in vitro through interaction with the Pro-rich region of Sec31A; ALG-2 increases recruitment of Sec23/24 and Sec13/31A to liposomes and mediates Sec13/31A binding to Sec23; EF-hand 1 Ca2+-binding site is required for this activity.","method":"In vitro COPII budding assay with semi-intact cells and liposomes, protein recruitment assay, EF-hand 1 mutation, pulldown","journal":"PLoS One","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution of COPII budding with defined mutants, multiple biochemical assays, single lab","pmids":["24069399"],"is_preprint":false},{"year":2013,"finding":"Nuclear ALG-2 interacts Ca2+-dependently with CHERP (Ca2+ homeostasis ER protein); Ca2+ mobilization recruits nuclear ALG-2 to CHERP-positive nuclear speckles; knockdown of CHERP or ALG-2 alters alternative splicing of IP3R1 pre-mRNA (inclusion of exons 41/42); CHERP binds IP3R1 RNA, indicating ALG-2 participates in nuclear pre-mRNA splicing regulation.","method":"Co-immunoprecipitation, live-cell time-lapse imaging, RNA immunoprecipitation, siRNA knockdown with RT-PCR splicing analysis","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP plus live imaging plus functional splicing readout plus RNA-IP, multiple orthogonal methods in one study","pmids":["24078636"],"is_preprint":false},{"year":2013,"finding":"VPS37B and VPS37C isoforms of ESCRT-I interact with ALG-2 more strongly than TSG101 does; ALG-2 functions as a Ca2+-dependent adaptor that bridges ALIX and ESCRT-I to form a ternary ESCRT-I/ALIX/ALG-2 complex, demonstrated with purified recombinant proteins.","method":"Far-Western blot with biotin-labeled ALG-2, pulldown assays, in vitro binding with purified recombinant ESCRT-I complexes and ALG-2","journal":"Bioscience, Biotechnology, and Biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro binding reconstitution plus Far-Western, single lab","pmids":["23924735"],"is_preprint":false},{"year":2014,"finding":"Luminal Ca2+ depletion decreases ER-to-Golgi transport rates; disruption of ALG-2/Sec31A interactions (via Pro-rich region mutations) causes severe ER-to-Golgi transport defects in intact cells; ALG-2/Sec31A interactions are required for stability of cargo receptor p24 and proper distribution of tethering protein p115, but not for Sec31A localization to ERES per se.","method":"In intact-cell transport assays, ultrastructural analysis (EM), Ca2+ depletion, dominant-negative ALG-2 binding domain disruption, p24/p115 distribution analysis","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple functional transport assays plus ultrastructural analysis plus pharmacological/genetic interventions, single lab","pmids":["25006245"],"is_preprint":false},{"year":2015,"finding":"Crystal structure of ALG-2 complexed with Sec31A peptide (type 2 motif PXPGF) shows the peptide binds to a third hydrophobic pocket (Pocket 3); Phe85 substitution abrogates Sec31A but not Alix binding; Tyr180 substitution eliminates Alix but not Sec31A binding, demonstrating that ALG-2 uses distinct hydrophobic surfaces to recognize type 1 (PPYPXYYY) vs type 2 (PXPGF) motifs.","method":"X-ray crystallography, single amino acid substitution mutagenesis, pulldown binding assays","journal":"International Journal of Molecular Sciences","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus systematic mutagenesis defining mechanistically distinct binding pockets, single lab","pmids":["25667979"],"is_preprint":false},{"year":2015,"finding":"Ca2+-dependent ALG-2 interaction with ALIX relieves the default intramolecular autoinhibitory interaction of ALIX, promoting CHMP4-dependent ALIX membrane association; EGFR activation increases ALG-2-ALIX interaction, and this is required for ALIX-mediated MVB sorting of activated EGFR; ALG-2 activation of ALIX does not affect cytokinetic abscission or EIAV budding.","method":"Pulldown assays, membrane fractionation, EGFR MVB sorting assay, co-transfection with dominant-negative ALIX mutants, EIAV budding assay","journal":"Cell Discovery","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple functional assays distinguishing pathway-specific ALG-2 effects, direct mechanism of ALIX activation demonstrated","pmids":["27462417"],"is_preprint":false},{"year":2016,"finding":"ALG-2 promotes ER exit site (ERES) localization and Ca2+-dependent polymerization of TFG (Trk-fused gene protein); ALG-2 interacts only with the ALG-2 homodimer (not ALG-2/peflin heterodimer); TFG deletion of the ALG-2-binding motif shortens TFG half-life at ERES; ALG-2 overexpression accumulates TFG at ERES.","method":"Co-immunoprecipitation, immunostaining, live-cell time-lapse imaging (thapsigargin-induced Ca2+ rise), in vitro cross-linking polymerization assay, motif deletion analysis","journal":"The FEBS Journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — live imaging plus in vitro polymerization assay plus deletion mutagenesis, multiple methods in one study","pmids":["27813252"],"is_preprint":false},{"year":2017,"finding":"ALG-2 interacts Ca2+-dependently with MISSL (MAPK1-interacting and spindle-stabilizing-like protein); MISSL and ALG-2 co-relocalize to puncta upon Ca2+ rise; knockdown of either MISSL or ALG-2 reduces secreted alkaline phosphatase secretion and delays ER-to-Golgi transport of procollagen I; MISSL and ALG-2 also interact with MAP1B, and MAP1B knockdown reverses reduced secretion caused by MISSL/ALG-2 depletion.","method":"Co-immunoprecipitation, live-cell imaging, siRNA knockdown with secretion assay (SEAP), procollagen I transport assay, epistasis by double knockdown","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP plus functional secretion assay plus genetic epistasis (double knockdown), multiple orthogonal methods","pmids":["28864773"],"is_preprint":false},{"year":2017,"finding":"ALG-2 interacts with HEBP2 (heme-binding protein 2); co-expression increases the cytoplasmic pool of ALG-2 and alters HEBP2 distribution; the ALG-2/HEBP2 complex affects mitotic spindle orientation/positioning and microtubule dynamics in cancer cells.","method":"Co-immunoprecipitation, subcellular localization imaging, mitotic spindle analysis, microtubule dynamics assay","journal":"Journal of Cellular Physiology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP plus imaging, limited mechanistic detail in abstract, single lab","pmids":["28004381"],"is_preprint":false},{"year":2018,"finding":"ALG-2 participates in plasma membrane repair; ALG-2 knockout DT-40 cells are more sensitive to electroporation; reintroduction of ALG-2 rescues this sensitivity; wild-type but not Ca2+-binding-deficient ALG-2 partially protects HeLa cells from digitonin-induced death; a peptide with the ALG-2 binding sequence of ALIX inhibits this protective function.","method":"PDCD6 gene disruption in DT-40 cells, electroporation survival assay, overexpression rescue, digitonin treatment, ALIX peptide competition","journal":"PLoS One","confidence":"High","confidence_rationale":"Tier 2 / Strong — gene knockout plus rescue plus peptide competition, Ca2+-binding requirement confirmed, multiple lines of evidence","pmids":["30240438"],"is_preprint":false},{"year":2018,"finding":"MAP1B interacts Ca2+-dependently with ALG-2 through a region lacking canonical ABM-1/ABM-2 motifs; MAP1B binding selectively competes with ABM-2-containing proteins (e.g., Sec31A) for ALG-2; MAP1B knockout cells show increased co-localization of ALG-2 with Sec31A; overexpression of wild-type MAP1B disperses ALG-2 and Sec31A distributions.","method":"Co-immunoprecipitation, point mutagenesis of MAP1B, MAP1B knockout cells, immunofluorescence co-localization","journal":"Biochemical and Biophysical Research Communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus KO cells plus imaging, single lab, multiple methods","pmids":["29432744"],"is_preprint":false},{"year":2020,"finding":"ALG-2 directly interacts with Rpn3 (a component of the 26S proteasome) and regulates proteasome activity upon Ca2+ elevation following T cell activation; this modulates MCL1 stability and accelerates T cell apoptosis (contraction); ALG-2 thus couples T cell activation to the subsequent apoptotic contraction phase.","method":"Co-immunoprecipitation (ALG-2-Rpn3), proteasome activity assay, MCL1 stability assay, T cell activation and apoptosis assays","journal":"Cell Death & Disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus functional assays for proteasome activity and MCL1 stability, single lab","pmids":["31919392"],"is_preprint":false},{"year":2020,"finding":"PDCD6 interacts with c-Raf via Co-IP, leading to activation of the c-Raf/MEK/ERK MAPK pathway and upregulation of MYC and JUN, promoting colorectal cancer cell proliferation.","method":"Co-immunoprecipitation, mass spectrometry, RNA-seq, Western blot of MAPK pathway components, in vitro and in vivo proliferation assays","journal":"Journal of Experimental & Clinical Cancer Research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP plus downstream signaling, limited mechanistic dissection of direct interaction, single lab","pmids":["32746883"],"is_preprint":false},{"year":2021,"finding":"ALG-2 and peflin together constitute a hetero-bifunctional COPII regulator; at steady-state Ca2+, ALG-2/peflin heterocomplexes bound to ERES confer a buffered secretion rate, while peflin-lacking ALG-2 complexes stimulate secretion; upon Ca2+ signaling, ALG-2-dependent effects on secretion can either increase or decrease ER export depending on signaling intensity; mechanistically, depression of secretion involves decreased COPII outer shell and increased peflin at ERES, while enhancement involves increased COPII outer shell and decreased peflin.","method":"ER-to-Golgi transport assays in NRK and PC12 cells, Ca2+ mobilization by ATP, COPII protein fractionation, peflin/ALG-2 siRNA knockdowns, secretion of physiological cargoes (collagen I, SEAP)","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — functional transport assay in two cell types, bidirectional Ca2+ effect, mechanistic dissection of heterodimer vs homodimer roles, multiple methods","pmids":["34762908"],"is_preprint":false},{"year":2021,"finding":"CDIP1 (cell death-inducing p53 target 1) interacts with ALG-2 Ca2+-dependently; ALG-2 promotes CDIP1 association with ESCRT-I (preferentially VPS37B/C-containing); co-expression of ALG-2 and ESCRT-I enhances CDIP1-induced caspase-3/7-mediated cell death; CDIP1 also binds VAPA/B via an FFAT-like motif.","method":"Co-immunoprecipitation, Ca2+-dependent pulldown, overexpression with caspase activity assay, domain deletion analysis","journal":"International Journal of Molecular Sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus functional cell death assay, single lab, multiple methods","pmids":["33503978"],"is_preprint":false},{"year":2022,"finding":"MAT2A interacts with PDCD6 and, upon AMPK activation, facilitates methylation of PDCD6 at K90, which increases PDCD6 protein stability; K90R mutation increases apoptosis and suppresses cervical cancer cell growth under glucose deprivation.","method":"Co-immunoprecipitation, immunoblotting, mass spectrometry, AMPK pathway inhibitors, K90R point mutation, cell viability and apoptosis assays","journal":"Cell Death Discovery","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus MS identification of methylation site plus mutagenesis with functional readout, single lab","pmids":["35396512"],"is_preprint":false},{"year":2024,"finding":"ALG-2 binds directly to acidic membranes in a Ca2+-dependent manner via electrostatic and hydrophobic interactions; charge-reversed mutants disrupt membrane recruitment; membrane binding is required for ERES localization but ESCRT-I binding can rescue membrane-binding-defective ALG-2 at lysosomes; Ca2+-dependent membrane binding and protein binding act together in cellular ALG-2 functions.","method":"Giant unilamellar vesicle (GUV) binding experiments, molecular dynamics simulations, charge-reversed mutagenesis, fluorescence imaging in cells (thapsigargin and lysosomal Ca2+ release), in vitro reconstitution with ESCRT-I","journal":"Proceedings of the National Academy of Sciences USA","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution plus MD simulations plus mutagenesis plus cellular imaging, multiple orthogonal methods","pmids":["38386713"],"is_preprint":false},{"year":2024,"finding":"ALG-2, upon lysosomal Ca2+ release (e.g., GPN-induced osmotic stress or TRPML1 activation), redistributes onto lysosomes and recruits ESCRT proteins, enhancing lysosomal membrane resilience to osmotic rupture; the ALG-2(ΔGF122) splice variant defective in ESCRT binding does not confer this protection; chelating cytoplasmic Ca2+ sensitizes lysosomes to rupture.","method":"Lysosomal leakage/rupture assays (sensitive fluorescent reporters), Ca2+ chelation (BAPTA), GPN and TRPML1 agonist treatments, ALG-2 and ΔGF122 overexpression, ESCRT recruitment imaging","journal":"Proceedings of the National Academy of Sciences USA","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple functional assays of lysosomal integrity, isoform control, Ca2+ manipulation, ESCRT recruitment, single lab but rigorous multi-method design","pmids":["38781205"],"is_preprint":false},{"year":2024,"finding":"PDCD6 interacts with LDHA and downregulates lactate metabolism; PDCD6 deficiency increases LDHA activity and lactate production, leading to RUBCN lactylation at K33, which promotes RUBCN interaction with VPS34, LAP (LC3-associated phagocytosis) formation, and bactericidal activity.","method":"Co-immunoprecipitation (PDCD6-LDHA), genetic knockout in mice and macrophages, LDHA pharmacological inhibition, lactate measurement, RUBCN lactylation site identification, LAP assays, bacterial killing assays","journal":"Nature Communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP plus KO mouse model plus biochemical pathway dissection including PTM identification, multiple methods in one study","pmids":["39578445"],"is_preprint":false},{"year":2005,"finding":"PDCD6 interacts with death-associated protein kinase 1 (DAPk1); co-transfection of PDCD6 and DAPk1 additively accelerates apoptosis via a caspase-3 dependent pathway.","method":"Yeast two-hybrid screening of human ovary cDNA library, co-transfection apoptosis assay with caspase-3 readout","journal":"Biotechnology Letters","confidence":"Low","confidence_rationale":"Tier 3 / Weak — yeast two-hybrid only for interaction, single cell line, single lab","pmids":["16132846"],"is_preprint":false},{"year":2008,"finding":"ALG-2 knockdown in HeLa cells causes G2/M cell cycle arrest and increased early apoptosis/cell death; pan-caspase inhibitor zVAD-fmk attenuates the increase in dead cells, indicating ALG-2 has an anti-apoptotic function in HeLa cells by facilitating G2/M checkpoint passage.","method":"siRNA knockdown, cell cycle analysis by flow cytometry, cell death quantification, caspase inhibitor (zVAD-fmk) rescue","journal":"Biochemical and Biophysical Research Communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA loss-of-function with cell cycle and death readouts plus pharmacological rescue, single lab","pmids":["19013425"],"is_preprint":false},{"year":2020,"finding":"ALG-2 interacts Ca2+-dependently with SARAF (a negative regulator of store-operated Ca2+ entry); ALG-2 overexpression interferes with NEDD4-family E3 ligase-mediated ubiquitination of SARAF at PPXY motifs proximal to the ALG-2 binding site, stabilizing SARAF; ALG-2 dimer promotes Ca2+-dependent SARAF CytD-to-CytD interactions.","method":"Semi-quantitative in vitro binding assay, pulldown with ubiquitination assay, half-life analysis, Strep-tag pulldown, Lys-to-Arg substitution mutants","journal":"International Journal of Molecular Sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — binding assay plus functional ubiquitination/stability analysis with mutagenesis, single lab","pmids":["32878247"],"is_preprint":false},{"year":2021,"finding":"PDCD6 interacts with the intracellular domain of cell adhesion molecule CHL1 in a Ca2+-dependent manner (Ca2+ chelation with BAPTA-AM abolishes association); a cell-penetrating CHL1-ICD peptide inhibits both the CHL1-PDCD6 association and PDCD6/CHL1-triggered neuronal survival.","method":"Co-immunoprecipitation, GST pull-down, proximity ligation assay in mouse brain tissue and cultured neurons, BAPTA-AM Ca2+ chelation, cell-penetrating peptide inhibition","journal":"FASEB BioAdvances","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus PLA in tissue plus functional peptide inhibition, single lab","pmids":["35024572"],"is_preprint":false},{"year":2012,"finding":"PATL1 (P-body component Pat1b) is a novel ALG-2-interacting protein; endogenous PATL1 and ALG-2 co-immunoprecipitate; a subset of ALG-2 co-localizes with PATL1 and the P-body marker DCP1A, identifying ALG-2 as having a potential role at P-bodies.","method":"In silico ABM screening, Far-Western blot, co-immunoprecipitation with endogenous proteins, immunofluorescence co-localization","journal":"Journal of Biochemistry","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP plus co-localization only, no functional consequence established, single lab","pmids":["22437941"],"is_preprint":false}],"current_model":"PDCD6/ALG-2 is a penta-EF-hand Ca2+-binding protein that acts as a multifunctional Ca2+-dependent adaptor: upon Ca2+ binding, an arginine-switch mechanism (Arg125, driven by Ca2+ at EF3) opens hydrophobic pockets that recognize Pro-rich motifs (type 1: PPYPXYYY in ALIX/TSG101; type 2: PXPGF in Sec31A) in diverse partners, enabling ALG-2 homodimers to bridge ALIX and ESCRT-I on endosomes to promote MVB sorting and lysosomal membrane repair, to stabilize the COPII outer coat protein Sec31A at ER exit sites to regulate ER-to-Golgi vesicular transport, to polymerize TFG and recruit MISSL/MAP1B at ERES, to shuttle to the nucleus with RBM22 and regulate alternative splicing of IP3R1 pre-mRNA via CHERP, to interact with the 26S proteasome subunit Rpn3 and modulate MCL1 stability during T cell contraction, to downregulate lactate metabolism via LDHA thereby controlling RUBCN lactylation and LC3-associated phagocytosis, and to participate in plasma membrane repair—all in a Ca2+-dependent and dimerization-dependent fashion, explaining its original identification as a required mediator of Ca2+-regulated apoptosis acting downstream of caspases."},"narrative":{"mechanistic_narrative":"PDCD6/ALG-2 is a penta-EF-hand Ca2+-binding protein that functions as a Ca2+-sensing molecular adaptor coupling intracellular Ca2+ transients to membrane traffic, membrane repair, and cell-fate decisions [PMID:8560270, PMID:11525164]. Ca2+ binding at its high-affinity EF-hands triggers a conformational change that exposes a hydrophobic surface [PMID:9832622, PMID:10360947], and crystallographic work resolved this as an arginine-switch in which Ca2+ loading at EF3 repositions Arg125 to open hydrophobic pockets that capture Pro-rich peptide motifs in partner proteins [PMID:18940611]; distinct pockets recognize type 1 (PPYP, as in ALIX/TSG101) versus type 2 (PXPGF, as in Sec31A) motifs, and the alternatively spliced ALG-2(ΔGF122) isoform reshapes these pockets to alter target selectivity [PMID:18256029, PMID:20691033, PMID:25667979]. Functional ALG-2 acts as a homodimer (mediated by the fifth EF-hand) and additionally binds acidic membranes Ca2+-dependently, with membrane and peptide engagement acting together for recruitment [PMID:10360947, PMID:38386713]. As an adaptor it bridges ALIX and ESCRT-I, relieves ALIX autoinhibition to promote CHMP4-dependent membrane association, and thereby drives MVB sorting of activated EGFR and recruits ESCRT to reinforce lysosomal membranes against rupture [PMID:19520058, PMID:27462417, PMID:38781205]. At ER exit sites ALG-2 binds and stabilizes the COPII outer-coat protein Sec31A and, together with its paralog peflin, constitutes a hetero-bifunctional Ca2+-tunable regulator of ER-to-Golgi secretion, also polymerizing TFG and recruiting MISSL/MAP1B [PMID:16957052, PMID:17196169, PMID:25006245, PMID:27813252, PMID:28864773, PMID:34762908]. In the nucleus ALG-2 partners with CHERP to regulate alternative splicing of IP3R1 pre-mRNA [PMID:24078636]. ALG-2 was originally identified as a required mediator of Ca2+-regulated apoptosis acting downstream of caspase activation [PMID:8560270, PMID:9164928], and it further couples Ca2+ signals to protein turnover and metabolism by engaging the proteasome subunit Rpn3 to control MCL1 during T-cell contraction and by restraining LDHA-driven lactate production that governs RUBCN lactylation and LC3-associated phagocytosis [PMID:31919392, PMID:39578445].","teleology":[{"year":1996,"claim":"Established that ALG-2 is a required component of Ca2+-regulated programmed cell death, defining the gene's founding biological role.","evidence":"Death-trap functional selection and antisense depletion in T cell hybridoma cells across TCR, Fas, and glucocorticoid death stimuli","pmids":["8560270"],"confidence":"High","gaps":["Molecular mechanism by which ALG-2 enables death not defined","No direct partners identified at this stage"]},{"year":1997,"claim":"Placed ALG-2 at a post-caspase step in apoptosis, distinguishing its function from upstream protease activation.","evidence":"Caspase activity and PARP cleavage assays in ALG-2-depleted clones showing normal cleavage yet death protection","pmids":["9164928"],"confidence":"High","gaps":["The downstream effectors ALG-2 acts through were not identified","Did not link the apoptotic role to a biochemical activity"]},{"year":1999,"claim":"Defined the Ca2+-dependent conformational logic and dimerization of ALG-2, showing Ca2+ binding exposes a hydrophobic surface and EF5 drives homodimerization.","evidence":"TNS fluorescence, gel filtration, cross-linking, CD and EF-hand mutagenesis on recombinant protein","pmids":["9832622","10360947"],"confidence":"High","gaps":["No physiological binding partner mapped to the exposed surface yet","Cellular consequence of the conformational change unresolved"]},{"year":1999,"claim":"Identified ALIX/AIP1 as the first strictly Ca2+-dependent ALG-2 partner cooperating in cell death, linking the conformational switch to a functional interaction.","evidence":"Yeast two-hybrid, reciprocal Co-IP, co-localization, and overexpression rescue, confirmed by a parallel study","pmids":["9880530","10200558"],"confidence":"High","gaps":["Structural basis of the interaction not yet resolved","Direct molecular role of the ALG-2-ALIX complex undefined"]},{"year":2002,"claim":"Generalized ALG-2 target recognition to a Pro/Gly/Tyr-rich binding mode by reconstituting direct nanomolar Ca2+-dependent binding to annexin VII/XI N-termini.","evidence":"SPR kinetics, GST pull-down, and biotin-ALG-2 overlay with recombinant annexins","pmids":["11883939","12445460"],"confidence":"High","gaps":["Functional outcome of annexin binding in cells not established","Did not yet define the precise peptide motif consensus"]},{"year":2001,"claim":"Resolved the first Ca2+-loaded ALG-2 crystal structure, showing a hinge rotation at EF3 that exposes a peptide-accepting cleft at the dimer interface.","evidence":"X-ray crystallography at 2.3 Å of Ca2+-bound ALG-2","pmids":["11525164"],"confidence":"High","gaps":["Did not capture a bound target peptide","Residue-level switch mechanism not yet defined"]},{"year":2005,"claim":"Extended ALG-2 into the ESCRT machinery by showing direct Ca2+-dependent binding to TSG101 and Ca2+-dependent endosomal recruitment.","evidence":"GST pull-down, yeast two-hybrid, overlay, and immunofluorescence with BAPTA-AM chelation in cells","pmids":["16004603"],"confidence":"High","gaps":["Whether ALG-2 bridges ALIX and ESCRT-I not yet tested","Functional consequence for endosomal sorting undefined"]},{"year":2006,"claim":"Connected ALG-2 to the early secretory pathway by demonstrating Ca2+-dependent recruitment to ER exit sites via Sec31A and reciprocal stabilization of Sec31A.","evidence":"Ca2+-dependent pull-down, overlay, RNAi, confocal imaging and pharmacological Ca2+ manipulation, independently replicated","pmids":["16957052","17196169"],"confidence":"High","gaps":["Effect on actual cargo transport rates not yet measured","Distinct binding mode versus ALIX/TSG101 not yet resolved"]},{"year":2006,"claim":"Placed ALG-2 in the nucleus through RBM22-mediated import, hinting at a role beyond membrane traffic.","evidence":"Yeast two-hybrid and confocal co-localization in NIH 3T3 cells and zebrafish embryos","pmids":["17045351"],"confidence":"Medium","gaps":["No direct binding reconstitution","Nuclear function not established at this stage"]},{"year":2008,"claim":"Defined the residue-level arginine-switch mechanism, showing Ca2+/EF3 repositions Arg125 to open the primary hydrophobic pocket that captures the ALIX PPYP motif.","evidence":"Multiple crystal structures including the ALG-2/Alix peptide complex plus mutagenesis-validated binding","pmids":["18940611"],"confidence":"High","gaps":["Did not resolve type 2 motif recognition","Membrane contribution to recruitment not addressed"]},{"year":2008,"claim":"Resolved a second, distinct ALG-2 binding pocket and motif class, explaining how the protein discriminates type 1 (PPYP) from type 2 (PXPGF) ligands.","evidence":"SPR, overlay, GST pull-down and mutagenesis on PLSCR3 ABS-1/ABS-2 sites","pmids":["18256029"],"confidence":"High","gaps":["Cellular role of PLSCR3 binding undefined","Structural detail of the second pocket awaited a co-crystal"]},{"year":2009,"claim":"Established ALG-2 as a Ca2+-dependent dimeric adaptor that physically bridges ALIX and TSG101, unifying its ESCRT-associated functions.","evidence":"Knockdown plus recombinant add-back ternary-complex reconstitution with isoform and dimerization mutant controls","pmids":["19520058"],"confidence":"High","gaps":["Membrane context of bridging not addressed","Downstream sorting cargo not yet defined in this study"]},{"year":2010,"claim":"Resolved the structural basis of isoform-selective target recognition, showing ΔGF122 repositions Arg125 to block Pocket 1 and differentially affect partner binding.","evidence":"Crystal structure of ALG-2(ΔGF122) plus systematic mutant binding assays against Alix/TSG101/annexin A11","pmids":["20691033"],"confidence":"High","gaps":["Physiological role of isoform switching not established","Did not capture the type 2 pocket structurally"]},{"year":2014,"claim":"Demonstrated that ALG-2/Sec31A engagement functionally controls ER-to-Golgi transport and cargo receptor stability, not merely Sec31A localization.","evidence":"Intact-cell transport assays, EM, Ca2+ depletion, and binding-domain disruption with p24/p115 analysis","pmids":["25006245","24069399"],"confidence":"High","gaps":["Direction of regulation (stimulatory vs inhibitory) not fully reconciled","Role of peflin not yet integrated"]},{"year":2013,"claim":"Assigned ALG-2 a nuclear function in alternative splicing, linking Ca2+ signaling to IP3R1 pre-mRNA processing via CHERP.","evidence":"Co-IP, live imaging, RNA-IP, and siRNA knockdown with RT-PCR splicing analysis","pmids":["24078636"],"confidence":"High","gaps":["Breadth of ALG-2-dependent splicing targets unknown","Mechanism of ALG-2 within the spliceosome undefined"]},{"year":2015,"claim":"Resolved the third hydrophobic pocket recognizing the Sec31A type 2 PXPGF motif and validated pocket-specific mutations separating Sec31A from Alix binding.","evidence":"Crystallography of the ALG-2/Sec31A peptide complex plus single-residue substitution binding assays","pmids":["25667979"],"confidence":"High","gaps":["In vivo significance of pocket separation not tested","Did not address membrane-binding contribution"]},{"year":2016,"claim":"Established that ALG-2 functions selectively as a homodimer at ERES to polymerize TFG and recruit MISSL/MAP1B, expanding its ERES regulatory repertoire.","evidence":"Co-IP, live imaging, in vitro polymerization assays, and double-knockdown epistasis with secretion readouts","pmids":["27813252","28864773","29432744"],"confidence":"High","gaps":["How homodimer vs heterodimer selection is regulated in cells unclear","Competition between MAP1B and Sec31A binding not fully mapped physiologically"]},{"year":2016,"claim":"Defined the molecular consequence of ALG-2-ALIX binding as relief of ALIX autoinhibition driving pathway-specific MVB sorting of activated EGFR.","evidence":"Pull-down, membrane fractionation, EGFR MVB sorting, and dominant-negative ALIX assays distinguishing pathway specificity","pmids":["27462417"],"confidence":"High","gaps":["Why ALG-2 affects MVB sorting but not abscission/budding not fully explained","In vivo relevance not established"]},{"year":2020,"claim":"Linked ALG-2 to regulated protein turnover by showing direct Rpn3/proteasome engagement that controls MCL1 stability during T-cell contraction.","evidence":"Co-IP, proteasome activity assays, and MCL1 stability with T-cell activation/apoptosis readouts","pmids":["31919392"],"confidence":"Medium","gaps":["Whether Rpn3 binding is direct via the canonical pockets unresolved","Reciprocal validation of proteasome regulation limited to single lab"]},{"year":2021,"claim":"Integrated ALG-2 with peflin into a hetero-bifunctional COPII regulator that bidirectionally tunes ER export according to Ca2+ signal intensity.","evidence":"ER-to-Golgi transport assays in two cell types with COPII fractionation and ALG-2/peflin knockdowns","pmids":["34762908"],"confidence":"High","gaps":["Quantitative threshold setting between buffering and stimulation undefined","Structural basis of heterodimer COPII effect not resolved"]},{"year":2024,"claim":"Demonstrated that ALG-2 directly engages acidic membranes Ca2+-dependently and that membrane binding and ESCRT-I binding act together for compartment-specific recruitment.","evidence":"GUV binding, molecular dynamics, charge-reversal mutagenesis, and cellular imaging with in vitro ESCRT-I reconstitution","pmids":["38386713"],"confidence":"High","gaps":["Relative contribution of membrane vs peptide binding across partners not quantified","Lipid specificity determinants only partly defined"]},{"year":2024,"claim":"Assigned ALG-2 a protective role in lysosomal membrane repair via Ca2+-triggered ESCRT recruitment, with the ESCRT-binding-defective ΔGF122 isoform unable to confer protection.","evidence":"Lysosomal rupture reporters, Ca2+ chelation, GPN/TRPML1 agonists, and isoform-controlled ESCRT recruitment imaging","pmids":["38781205"],"confidence":"High","gaps":["In vivo physiological setting of lysosomal repair not tested","Crosstalk with plasma-membrane repair function not delineated"]},{"year":2024,"claim":"Connected PDCD6 to immunometabolism by showing it restrains LDHA-driven lactate production, controlling RUBCN lactylation and LC3-associated phagocytosis.","evidence":"Co-IP, knockout mice/macrophages, LDHA inhibition, lactylation site mapping, and bactericidal LAP assays","pmids":["39578445"],"confidence":"High","gaps":["Whether PDCD6-LDHA binding uses the canonical Ca2+/peptide mechanism unclear","Ca2+ dependence of this metabolic axis not addressed"]},{"year":null,"claim":"How the diverse ALG-2 functions—secretory traffic, ESCRT-dependent membrane repair, nuclear splicing, proteasome and metabolic regulation—are coordinated and prioritized by distinct Ca2+ signal patterns within a single cell remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No integrated model of partner selection under physiological Ca2+ dynamics","In vivo phenotypes tying the molecular activities to organismal physiology largely lacking","Mechanistic basis for several Co-IP-only partners (c-Raf, VEGFR-2, DAPk1, PATL1) not established"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[22,29,32]},{"term_id":"GO:0140299","term_label":"molecular sensor activity","supporting_discovery_ids":[2,6,15,19]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[43]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[14,33,48]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[3,7]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[13,32]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[14,30,33]},{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[43,44]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[7,16,28]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[36,43]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[14,30,32,40]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[0,1,38]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[36,44]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[28]}],"complexes":["ESCRT-I/ALIX/ALG-2 ternary complex","ALG-2 homodimer","ALG-2/peflin heterodimer","COPII outer coat (Sec13/31A)"],"partners":["PDCD6IP/ALIX","TSG101","SEC31A","PEF1","ANXA11","TFG","CHERP","PSMD3"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O75340","full_name":"Programmed cell death protein 6","aliases":["Apoptosis-linked gene 2 protein homolog","ALG-2"],"length_aa":191,"mass_kda":21.9,"function":"Calcium sensor that plays a key role in processes such as endoplasmic reticulum (ER)-Golgi vesicular transport, endosomal biogenesis or membrane repair. Acts as an adapter that bridges unrelated proteins or stabilizes weak protein-protein complexes in response to calcium: calcium-binding triggers exposure of apolar surface, promoting interaction with different sets of proteins thanks to 3 different hydrophobic pockets, leading to translocation to membranes (PubMed:20691033, PubMed:25667979). Involved in ER-Golgi transport by promoting the association between PDCD6IP and TSG101, thereby bridging together the ESCRT-III and ESCRT-I complexes (PubMed:19520058). Together with PEF1, acts as a calcium-dependent adapter for the BCR(KLHL12) complex, a complex involved in ER-Golgi transport by regulating the size of COPII coats (PubMed:27716508). In response to cytosolic calcium increase, the heterodimer formed with PEF1 interacts with, and bridges together the BCR(KLHL12) complex and SEC31 (SEC31A or SEC31B), promoting monoubiquitination of SEC31 and subsequent collagen export, which is required for neural crest specification (PubMed:27716508). Involved in the regulation of the distribution and function of MCOLN1 in the endosomal pathway (PubMed:19864416). Promotes localization and polymerization of TFG at endoplasmic reticulum exit site (PubMed:27813252). Required for T-cell receptor-, Fas-, and glucocorticoid-induced apoptosis (By similarity). May mediate Ca(2+)-regulated signals along the death pathway: interaction with DAPK1 can accelerate apoptotic cell death by increasing caspase-3 activity (PubMed:16132846). Its role in apoptosis may however be indirect, as suggested by knockout experiments (By similarity). May inhibit KDR/VEGFR2-dependent angiogenesis; the function involves inhibition of VEGF-induced phosphorylation of the Akt signaling pathway (PubMed:21893193). In case of infection by HIV-1 virus, indirectly inhibits HIV-1 production by affecting viral Gag expression and distribution (PubMed:27784779) Has a lower Ca(2+) affinity than isoform 1 (By similarity)","subcellular_location":"Endoplasmic reticulum membrane; Cytoplasmic vesicle, COPII-coated vesicle membrane; Cytoplasm; Nucleus; Endosome","url":"https://www.uniprot.org/uniprotkb/O75340/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PDCD6","classification":"Not Classified","n_dependent_lines":346,"n_total_lines":1208,"dependency_fraction":0.28642384105960267},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000249915","cell_line_id":"CID000994","localizations":[{"compartment":"cytoplasmic","grade":3},{"compartment":"nucleoplasm","grade":3}],"interactors":[{"gene":"HDAC3","stoichiometry":10.0},{"gene":"SEC23IP","stoichiometry":10.0},{"gene":"SEC31A","stoichiometry":4.0},{"gene":"PDCD6IP","stoichiometry":0.2},{"gene":"PEF1","stoichiometry":0.2},{"gene":"HEBP2","stoichiometry":0.2},{"gene":"NEDD8;NEDD8-MDP1","stoichiometry":0.2},{"gene":"IST1","stoichiometry":0.2},{"gene":"SEC13","stoichiometry":0.2},{"gene":"MAP1B","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000994","total_profiled":1310},"omim":[{"mim_id":"618695","title":"CILIARY DYSKINESIA, PRIMARY, 42; CILD42","url":"https://www.omim.org/entry/618695"},{"mim_id":"614086","title":"MULTICILIATE DIFFERENTIATION AND DNA SYNTHESIS-ASSOCIATED CELL CYCLE PROTEIN; MCIDAS","url":"https://www.omim.org/entry/614086"},{"mim_id":"610033","title":"PENTA-EF-HAND DOMAIN-CONTAINING PROTEIN 1; PEF1","url":"https://www.omim.org/entry/610033"},{"mim_id":"608074","title":"PROGRAMMED CELL DEATH 6-INTERACTING PROTEIN; PDCD6IP","url":"https://www.omim.org/entry/608074"},{"mim_id":"601057","title":"PROGRAMMED CELL DEATH 6; PDCD6","url":"https://www.omim.org/entry/601057"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"},{"location":"Nucleoplasm","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PDCD6"},"hgnc":{"alias_symbol":["ALG-2","PEF1B"],"prev_symbol":[]},"alphafold":{"accession":"O75340","domains":[{"cath_id":"1.10.238.10","chopping":"24-188","consensus_level":"medium","plddt":94.7142,"start":24,"end":188}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O75340","model_url":"https://alphafold.ebi.ac.uk/files/AF-O75340-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O75340-F1-predicted_aligned_error_v6.png","plddt_mean":89.12},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PDCD6","jax_strain_url":"https://www.jax.org/strain/search?query=PDCD6"},"sequence":{"accession":"O75340","fasta_url":"https://rest.uniprot.org/uniprotkb/O75340.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O75340/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O75340"}},"corpus_meta":[{"pmid":"8560270","id":"PMC_8560270","title":"Interfering with apoptosis: Ca(2+)-binding protein ALG-2 and Alzheimer's disease gene ALG-3.","date":"1996","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/8560270","citation_count":451,"is_preprint":false},{"pmid":"10200558","id":"PMC_10200558","title":"Alix, a novel mouse protein undergoing calcium-dependent interaction with the apoptosis-linked-gene 2 (ALG-2) protein.","date":"1999","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/10200558","citation_count":215,"is_preprint":false},{"pmid":"9880530","id":"PMC_9880530","title":"Cloning of AIP1, a novel protein that associates with the apoptosis-linked gene ALG-2 in a Ca2+-dependent reaction.","date":"1999","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/9880530","citation_count":201,"is_preprint":false},{"pmid":"12860994","id":"PMC_12860994","title":"The ALG-2-interacting protein Alix associates with CHMP4b, a human homologue of yeast Snf7 that is involved in 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journal","url":"https://pubmed.ncbi.nlm.nih.gov/20670214","citation_count":10,"is_preprint":false},{"pmid":"32878247","id":"PMC_32878247","title":"The Penta-EF-Hand ALG-2 Protein Interacts with the Cytosolic Domain of the SOCE Regulator SARAF and Interferes with Ubiquitination.","date":"2020","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/32878247","citation_count":9,"is_preprint":false},{"pmid":"23649269","id":"PMC_23649269","title":"Mammalian ESCRT-III-related protein IST1 has a distinctive met-pro repeat sequence that is essential for interaction with ALG-2 in the presence of Ca2+.","date":"2013","source":"Bioscience, biotechnology, and biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/23649269","citation_count":9,"is_preprint":false},{"pmid":"30259798","id":"PMC_30259798","title":"Adaptor functions of the Ca2+-binding protein ALG-2 in protein transport from the endoplasmic reticulum.","date":"2018","source":"Bioscience, biotechnology, and biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/30259798","citation_count":8,"is_preprint":false},{"pmid":"23137875","id":"PMC_23137875","title":"Association between two single nucleotide polymorphisms of PDCD6 gene and increased endometriosis risk.","date":"2012","source":"Human immunology","url":"https://pubmed.ncbi.nlm.nih.gov/23137875","citation_count":8,"is_preprint":false},{"pmid":"34723149","id":"PMC_34723149","title":"The Argonaute Proteins ALG-1 and ALG-2 Are Linked to Stress Resistance and Proteostasis.","date":"2021","source":"microPublication biology","url":"https://pubmed.ncbi.nlm.nih.gov/34723149","citation_count":8,"is_preprint":false},{"pmid":"36132985","id":"PMC_36132985","title":"Electroacupuncture-Regulated miR-34a-3p/PDCD6 Axis Promotes Post-Spinal Cord Injury Recovery in Both In Vitro and In Vivo Settings.","date":"2022","source":"Journal of immunology research","url":"https://pubmed.ncbi.nlm.nih.gov/36132985","citation_count":8,"is_preprint":false},{"pmid":"31814542","id":"PMC_31814542","title":"Structures and functions of penta-EF-hand calcium-binding proteins and their interacting partners: enigmatic relationships between ALG-2 and calpain-7.","date":"2019","source":"Bioscience, biotechnology, and biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/31814542","citation_count":8,"is_preprint":false},{"pmid":"29432744","id":"PMC_29432744","title":"A microtubule-associated protein MAP1B binds to and regulates localization of a calcium-binding protein ALG-2.","date":"2018","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/29432744","citation_count":8,"is_preprint":false},{"pmid":"22641034","id":"PMC_22641034","title":"Structural recognition mechanisms between human Src homology domain 3 (SH3) and ALG-2-interacting protein X (Alix).","date":"2012","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/22641034","citation_count":8,"is_preprint":false},{"pmid":"22837710","id":"PMC_22837710","title":"Prediction of a new ligand-binding site for type 2 motif based on the crystal structure of ALG-2 by dry and wet approaches.","date":"2012","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/22837710","citation_count":8,"is_preprint":false},{"pmid":"32597292","id":"PMC_32597292","title":"miR-9875 functions in antiviral immunity by targeting PDCD6 in mud crab (Scylla paramamosain).","date":"2020","source":"Virulence","url":"https://pubmed.ncbi.nlm.nih.gov/32597292","citation_count":7,"is_preprint":false},{"pmid":"35024572","id":"PMC_35024572","title":"Interplay in neural functions of cell adhesion molecule close homolog of L1 (CHL1) and Programmed Cell Death 6 (PDCD6).","date":"2021","source":"FASEB bioAdvances","url":"https://pubmed.ncbi.nlm.nih.gov/35024572","citation_count":7,"is_preprint":false},{"pmid":"11804327","id":"PMC_11804327","title":"Mapping of the interaction sites between apoptosis linked gene ALG-2 and HEED.","date":"2001","source":"Molecules and cells","url":"https://pubmed.ncbi.nlm.nih.gov/11804327","citation_count":7,"is_preprint":false},{"pmid":"23296612","id":"PMC_23296612","title":"Biochemical and immunological detection of physical interactions between penta-EF-hand protein ALG-2 and its binding partners.","date":"2013","source":"Methods in molecular biology (Clifton, N.J.)","url":"https://pubmed.ncbi.nlm.nih.gov/23296612","citation_count":7,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":47985,"output_tokens":12884,"usd":0.168607,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":24635,"output_tokens":6749,"usd":0.14595,"stage2_stop_reason":"end_turn"},"total_usd":0.314557,"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\": 1996,\n      \"finding\": \"ALG-2 (PDCD6) encodes a Ca2+-binding protein required for T cell receptor-, Fas-, and glucocorticoid-induced apoptosis; ALG-2-depleted 3DO T cell hybridoma cells are protected from these death stimuli, placing ALG-2 as a required mediator of Ca2+-regulated apoptotic signaling.\",\n      \"method\": \"Functional selection (death trap assay), antisense depletion of ALG-2 with loss-of-function apoptosis phenotype\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — foundational loss-of-function study with clear apoptosis phenotype, replicated across multiple death stimuli, widely cited and independently confirmed\",\n      \"pmids\": [\"8560270\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"ALG-2 functions downstream of or independently of ICE/Ced-3 (caspase) activation during apoptosis; ALG-2-depleted cells exhibit normal caspase-mediated PARP cleavage yet are protected from cell death, indicating ALG-2 acts at a post-caspase step.\",\n      \"method\": \"Caspase activity assay (fluorogenic substrate, PARP cleavage) in ALG-2-depleted T cell hybridoma clones\",\n      \"journal\": \"Journal of Immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct biochemical epistasis experiment using endogenous substrate cleavage assays, consistent with and extending the founding paper\",\n      \"pmids\": [\"9164928\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Ca2+ binding to ALG-2 induces exposure of a hydrophobic surface on the protein, as detected by a fluorescent hydrophobicity probe (TNS); this conformational change occurs with half-maximal effect at ~6 µM Ca2+, and Mg2+ is not effective.\",\n      \"method\": \"Fluorescence spectroscopy with hydrophobicity probe TNS; gel filtration; biochemical fractionation of mammalian cells\",\n      \"journal\": \"Journal of Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro biophysical characterization, single lab, but clear mechanistic result on Ca2+-dependent conformational change\",\n      \"pmids\": [\"9832622\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"ALG-2 interacts with AIP1/Alix in a strictly Ca2+-dependent manner; both proteins co-localize in the cytosol; overexpression of a truncated AIP1 protects cells from trophic factor withdrawal-induced death, indicating AIP1 cooperates with ALG-2 in the Ca2+-dependent cell death pathway.\",\n      \"method\": \"Yeast two-hybrid screening, co-immunoprecipitation, subcellular co-localization, overexpression rescue assay\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP plus functional rescue, independently confirmed by parallel study (PMID:10200558), multiple methods\",\n      \"pmids\": [\"9880530\", \"10200558\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"ALG-2 possesses two high-affinity Ca2+-binding sites; Ca2+ binding induces conformational changes in both N- and C-terminal domains; the fifth EF-hand participates in homodimerization; Ca2+ binding to both strong sites is required for Ca2+-induced protein aggregation.\",\n      \"method\": \"Recombinant protein expression, gel filtration, chemical cross-linking, fluorescence spectroscopy, circular dichroism, site-directed mutagenesis (Glu47Ala/Glu114Ala)\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with mutagenesis and multiple spectroscopic methods, single lab\",\n      \"pmids\": [\"10360947\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Two alternatively spliced isoforms of ALG-2 exist (ALG-2,5 full-length and ALG-2,1 lacking Gly121/Phe122); they differ in Ca2+ affinity; ALG-2,1 does not interact with AIP1/Alix despite otherwise similar properties, indicating these two residues are critical for target recognition.\",\n      \"method\": \"cDNA cloning, Ca2+ binding assays, yeast two-hybrid interaction assay\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid plus Ca2+ binding biochemistry, single lab, two orthogonal methods\",\n      \"pmids\": [\"10744743\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"X-ray crystal structure of Ca2+-loaded ALG-2 (des1-20, 2.3 Å) reveals five EF-hands folding into eight α-helices, dimer formation, Ca2+ binding to EF1, EF3, and EF5; a rigid-body rotation of EF1-2 relative to EF4-5 (hinge at EF3) upon Ca2+ loading exposes a hydrophobic patch and a large cleft near the dimer interface that accommodates a Gly/Pro-rich peptide.\",\n      \"method\": \"X-ray crystallography at 2.3 Å resolution; limited proteolysis for crystal preparation\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with mechanistic interpretation of Ca2+-dependent conformational change, foundational structural paper\",\n      \"pmids\": [\"11525164\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"ALG-2 forms a Ca2+-independent homodimer and a Ca2+-dependent heterodimer with its closest paralog peflin; peflin dissociates from ALG-2 in the presence of Ca2+; peflin translocates to membrane/cytoskeletal fractions in Ca2+ conditions while ALG-2 is also found in the nucleus.\",\n      \"method\": \"Co-immunoprecipitation with monoclonal anti-peflin antibody, epitope-tag co-IP, gel filtration, immunofluorescence, subcellular fractionation\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP plus gel filtration plus fractionation, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"11278427\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"ALG-2 interacts directly and Ca2+-dependently with the N-terminal domain of annexin XI; KD ~70 nM for the high-affinity site; the Pro/Gly/Tyr/Ala-rich hydrophobic region of annexin XI masks Ca2+-dependently exposed hydrophobic surface of ALG-2.\",\n      \"method\": \"Yeast two-hybrid, GST pull-down, biotin-ALG-2 overlay assay, surface plasmon resonance (SPR), fluorescence probe inhibition\",\n      \"journal\": \"Biochemical and Biophysical Research Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct binding reconstitution with SPR kinetics plus overlay, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"11883939\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"ALG-2 binds directly and Ca2+-dependently to the N-terminal domains of both annexin VII and annexin XI with two binding sites (KD ~40-60 nM high-affinity, ~500-700 nM low-affinity), establishing a common PGAYQ-biased binding mode.\",\n      \"method\": \"Biotin-ALG-2 overlay assay, surface plasmon resonance biosensor with GST-fusion proteins\",\n      \"journal\": \"Biochimica et Biophysica Acta\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with SPR kinetics and overlay, single lab, two orthogonal methods\",\n      \"pmids\": [\"12445460\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"The fifth EF-hand (EF-5) of both ALG-2 and peflin is essential for dimerization and protein stability; EF-5 deletion mutants are rapidly degraded by the proteasome.\",\n      \"method\": \"Deletion mutagenesis, pulse-chase experiment, proteasome inhibitor (MG132) treatment, Western blot, subcellular fractionation\",\n      \"journal\": \"Archives of Biochemistry and Biophysics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function mutagenesis with pharmacological rescue, single lab\",\n      \"pmids\": [\"11883899\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"ALG-2 interacts with ASK1 (apoptosis signal-regulating kinase 1) at its C-terminus (aa 941-1375) in a Ca2+-dependent manner; the ALG-2,1 isoform lacking Gly121/Phe122 does not bind ASK1; co-expression of ALG-2 redirects ASK1 to the nucleus and inhibits ASK1-induced JNK activation.\",\n      \"method\": \"Co-immunoprecipitation in BOSC23 cells, in vitro binding, co-transfection with JNK activity readout, subcellular localization by microscopy\",\n      \"journal\": \"FEBS Letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus functional JNK assay, isoform specificity demonstrated, single lab\",\n      \"pmids\": [\"12372597\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"ALG-2 interacts directly with Alix through a region (aa 794-827) containing four tandem PxY repeats; seven proline and four tyrosine residues in PxY repeats are critical for binding affinity; Ca2+-binding-deficient ALG-2(E47A/E114A) does not co-immunoprecipitate with Alix; ALG-2 is required for the subcellular punctate distribution of the Alix C-terminal half.\",\n      \"method\": \"Yeast two-hybrid, biotin-ALG-2 overlay, alanine-substitution mutagenesis, co-immunoprecipitation, GFP-fusion fluorescence microscopy\",\n      \"journal\": \"Journal of Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct binding reconstitution with mutagenesis plus Co-IP plus imaging, multiple orthogonal methods in one study\",\n      \"pmids\": [\"14999017\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"ALG-2 directly binds the proline-rich region (PRR) of TSG101 (ESCRT-I component) in a Ca2+-dependent manner; ALG-2 also associates with hVps28 and hVps37A indirectly via TSG101; ALG-2 co-localizes with SKD1(E235Q)-induced aberrant endosomes in a Ca2+-dependent manner; Ca2+ chelation abolishes this punctate endosomal localization.\",\n      \"method\": \"GST pull-down, yeast two-hybrid, biotin-ALG-2 overlay assay, immunofluorescence microscopy, Ca2+ chelator (BAPTA-AM) treatment\",\n      \"journal\": \"The Biochemical Journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct binding demonstrated by overlay plus pulldown plus yeast two-hybrid, subcellular localization with Ca2+ dependence, multiple methods\",\n      \"pmids\": [\"16004603\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"ALG-2 is recruited to ER exit sites (ERES) via Ca2+-dependent interaction with Sec31A (COPII outer coat component); ALG-2 in turn stabilizes Sec31A at ERES; Ca2+-binding-deficient ALG-2 mutant loses ERES localization; Ca2+ chelation disperses ALG-2 and reduces membrane-associated Sec31A.\",\n      \"method\": \"Ca2+-dependent GST pull-down, biotin-ALG-2 overlay, immunofluorescence confocal microscopy, RNAi knockdown, Ca2+ ionophore/chelator treatments, overexpression of Sec31A PRR as dominant negative\",\n      \"journal\": \"Molecular Biology of the Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (pulldown, overlay, RNAi, imaging, pharmacological), replicated by independent concurrent study (PMID:17196169)\",\n      \"pmids\": [\"16957052\", \"17196169\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"ALG-2 subcellular distribution oscillates between cytosol and punctate (COPII/Sec31A-positive) localization in phase with intracellular Ca2+ oscillations triggered by physiological stimuli (ATP, EGF, prostaglandin, histamine); a Ca2+-binding-deficient mutant does not redistribute.\",\n      \"method\": \"Live-cell fluorescence imaging of tagged ALG-2 simultaneous with Ca2+ indicators; Ca2+-binding-deficient mutant as control\",\n      \"journal\": \"Biochemical and Biophysical Research Communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live-cell imaging with Ca2+ co-monitoring and mutant control, single lab\",\n      \"pmids\": [\"17214967\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"ALG-2 is translocated to the nucleus upon co-expression with the RNA-binding protein RBM22, which shuttles between cytoplasm and nucleus; in zebrafish embryos the two proteins co-localize within the nucleus, suggesting RBM22-mediated nuclear import of ALG-2.\",\n      \"method\": \"Yeast two-hybrid screening, confocal microscopy of fluorescent fusions in NIH 3T3 cells and zebrafish embryos\",\n      \"journal\": \"Biochimica et Biophysica Acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-localization imaging plus yeast two-hybrid, two model systems, but no direct binding reconstitution or functional consequence established\",\n      \"pmids\": [\"17045351\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"POSH scaffold protein forms a Ca2+-dependent complex with ALG-2 and ALIX in Drosophila; overexpression of ALG-2 causes roughened/melanized eye phenotypes; co-overexpression with POSH enhances these phenotypes; ALG-2 overexpression induces ectopic JNK activation, suggesting POSH/ALG-2/ALIX function together in JNK pathway regulation.\",\n      \"method\": \"Co-immunoprecipitation in Drosophila cells, genetic overexpression in eye imaginal discs, JNK activity assay\",\n      \"journal\": \"FEBS Letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus in vivo genetic epistasis in Drosophila, single lab\",\n      \"pmids\": [\"16698022\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"ALG-2 binds to Scotin (a p53-inducible ER-membrane protein) in a strictly Ca2+-dependent manner; overexpression of ALG-2 increases Scotin protein levels, indicating ALG-2 stabilizes Scotin.\",\n      \"method\": \"In vitro binding assay (synthesized C-terminal Scotin fragment on immobilized ALG-2), co-immunoprecipitation in MCF7 and U2OS cell lines, overexpression Western blot\",\n      \"journal\": \"Archives of Biochemistry and Biophysics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus in vitro direct binding, multiple cell lines, Ca2+ dependence confirmed, single lab\",\n      \"pmids\": [\"17889823\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"X-ray crystal structures of Ca2+-free and Ca2+-bound ALG-2 and of the ALG-2/Alix799-814 peptide complex reveal a Ca2+/EF3-driven arginine switch: Ca2+ binding to EF3 repositions Arg125, opening a primary hydrophobic pocket (Pocket 1) that accepts the PPYP motif of Alix; in vitro binding assays with mutant proteins validate this mechanism.\",\n      \"method\": \"X-ray crystallography (multiple structures), in vitro binding assay with ALG-2 and Alix mutants\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple crystal structures plus mutagenesis-validated binding mechanism, mechanistically definitive\",\n      \"pmids\": [\"18940611\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"ALG-2 interacts with Alix and pro-caspase-8; Alix forms a complex with TNF-R1-containing endosomes in a manner dependent on ESCRT binding; deletion of the ALG-2-binding site on Alix significantly reduces TNF-R1-induced cell death without affecting receptor endocytosis, placing ALG-2 in the TNF-R1 death signaling pathway.\",\n      \"method\": \"Mass spectrometry of Alix co-immunoprecipitates, Co-IP, overexpression of Alix deletion mutants, cell death assays, motoneuron primary culture\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus domain deletion functional assay, single lab\",\n      \"pmids\": [\"18936101\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"ALG-2 interacts with PLSCR3 Ca2+-dependently via two distinct binding sites: ABS-1 (PPYP-type, type 1 motif, Alix-like) recognized only by full-length ALG-2, and ABS-2 (PXPGF-type, type 2 motif) recognized by both full-length ALG-2 and the ALG-2(ΔGF122) isoform; Phe49 in ABS-2 is critical for binding.\",\n      \"method\": \"Co-immunoprecipitation, GST pull-down, biotin-ALG-2 overlay, surface plasmon resonance with synthetic oligopeptides, mutagenesis\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — SPR kinetics plus mutagenesis plus multiple pulldown methods defining two distinct binding pockets, single lab\",\n      \"pmids\": [\"18256029\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"ALG-2 functions as a Ca2+-dependent adaptor protein that bridges Alix and TSG101: ALG-2 knockdown abolishes Alix-TSG101 association in pulldown assays; recombinant ALG-2 restores the interaction; the shorter ALG-2(ΔGF122) isoform and a dimerization-defective mutant cannot bridge the two proteins, indicating the ALG-2 dimer is required.\",\n      \"method\": \"Strep-tag pulldown assay with ALG-2 knockdown cells, add-back of purified recombinant ALG-2, isoform and dimerization mutant controls\",\n      \"journal\": \"Biochemical and Biophysical Research Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reconstitution of ternary complex with knockdown plus add-back, isoform controls, dimerization requirement demonstrated\",\n      \"pmids\": [\"19520058\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"ALG-2 binds directly to the NH2-terminal cytosolic tail of mucolipin-1 (MCOLN1) Ca2+-dependently; the interaction is mediated by residues 37-49 of MCOLN1; mutation of the ALG-2-binding domain in MCOLN1 reduces MCOLN1-induced accumulation of aberrant endosomes, indicating ALG-2 modulates MCOLN1 function in the late endosomal-lysosomal pathway.\",\n      \"method\": \"Co-immunoprecipitation, direct binding assay, deletion/point mutagenesis of MCOLN1, fluorescence microscopy with dominant-negative Vps4B\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct binding plus mutagenesis with functional endosomal phenotype, multiple methods, single lab\",\n      \"pmids\": [\"19864416\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The ALG-2-binding site (ABS) in Sec31A (aa 839-851 in Pro-rich region) controls retention kinetics of Sec31A at ERES; FRAP analysis shows ABS deletion reduces the high-affinity/slow-turnover population of Sec31A at ERES.\",\n      \"method\": \"Stable cell lines with GFP-ALG-2 and Sec31A-RFP, live-cell imaging after Ca2+ mobilization, biotin-ALG-2 overlay to map ABS, FRAP\",\n      \"journal\": \"Bioscience, Biotechnology, and Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live-cell FRAP plus overlay mapping, single lab, two orthogonal methods\",\n      \"pmids\": [\"20834162\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Crystal structure of ALG-2(ΔGF122) in Ca2+-bound form reveals that deletion of Gly121-Phe122 shortens α-helix 5 and repositions Arg125 to partially block Pocket 1, explaining failure to bind Alix; F122A/G substitutions (but not F122W) increase Alix-binding by expanding Pocket 2, while affecting binding to TSG101 and annexin A11 differently, demonstrating structural flexibility in target recognition.\",\n      \"method\": \"X-ray crystallography, in vitro binding assays with multiple ALG-2 point mutants and Alix/TSG101/annexin A11\",\n      \"journal\": \"BMC Structural Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus systematic mutagenesis binding assays, mechanistically definitive for isoform selectivity\",\n      \"pmids\": [\"20691033\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"PDCD6/ALG-2 directly binds VEGFR-2 and suppresses phosphorylation of PI3K/Akt/mTOR/GSK-3β/p70S6K signaling, reducing VEGF-induced endothelial proliferation, invasion, and tube formation in vitro.\",\n      \"method\": \"Co-immunoprecipitation to show VEGFR-2 binding, Western blot of downstream signaling, cell migration/invasion/tube formation assays with recombinant PDCD6\",\n      \"journal\": \"Cellular Signalling\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP plus signaling readouts, no direct binding reconstitution, single lab\",\n      \"pmids\": [\"21893193\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ALG-2/Ca2+ attenuates COPII vesicle budding in vitro through interaction with the Pro-rich region of Sec31A; ALG-2 increases recruitment of Sec23/24 and Sec13/31A to liposomes and mediates Sec13/31A binding to Sec23; EF-hand 1 Ca2+-binding site is required for this activity.\",\n      \"method\": \"In vitro COPII budding assay with semi-intact cells and liposomes, protein recruitment assay, EF-hand 1 mutation, pulldown\",\n      \"journal\": \"PLoS One\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution of COPII budding with defined mutants, multiple biochemical assays, single lab\",\n      \"pmids\": [\"24069399\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Nuclear ALG-2 interacts Ca2+-dependently with CHERP (Ca2+ homeostasis ER protein); Ca2+ mobilization recruits nuclear ALG-2 to CHERP-positive nuclear speckles; knockdown of CHERP or ALG-2 alters alternative splicing of IP3R1 pre-mRNA (inclusion of exons 41/42); CHERP binds IP3R1 RNA, indicating ALG-2 participates in nuclear pre-mRNA splicing regulation.\",\n      \"method\": \"Co-immunoprecipitation, live-cell time-lapse imaging, RNA immunoprecipitation, siRNA knockdown with RT-PCR splicing analysis\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP plus live imaging plus functional splicing readout plus RNA-IP, multiple orthogonal methods in one study\",\n      \"pmids\": [\"24078636\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"VPS37B and VPS37C isoforms of ESCRT-I interact with ALG-2 more strongly than TSG101 does; ALG-2 functions as a Ca2+-dependent adaptor that bridges ALIX and ESCRT-I to form a ternary ESCRT-I/ALIX/ALG-2 complex, demonstrated with purified recombinant proteins.\",\n      \"method\": \"Far-Western blot with biotin-labeled ALG-2, pulldown assays, in vitro binding with purified recombinant ESCRT-I complexes and ALG-2\",\n      \"journal\": \"Bioscience, Biotechnology, and Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro binding reconstitution plus Far-Western, single lab\",\n      \"pmids\": [\"23924735\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Luminal Ca2+ depletion decreases ER-to-Golgi transport rates; disruption of ALG-2/Sec31A interactions (via Pro-rich region mutations) causes severe ER-to-Golgi transport defects in intact cells; ALG-2/Sec31A interactions are required for stability of cargo receptor p24 and proper distribution of tethering protein p115, but not for Sec31A localization to ERES per se.\",\n      \"method\": \"In intact-cell transport assays, ultrastructural analysis (EM), Ca2+ depletion, dominant-negative ALG-2 binding domain disruption, p24/p115 distribution analysis\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple functional transport assays plus ultrastructural analysis plus pharmacological/genetic interventions, single lab\",\n      \"pmids\": [\"25006245\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Crystal structure of ALG-2 complexed with Sec31A peptide (type 2 motif PXPGF) shows the peptide binds to a third hydrophobic pocket (Pocket 3); Phe85 substitution abrogates Sec31A but not Alix binding; Tyr180 substitution eliminates Alix but not Sec31A binding, demonstrating that ALG-2 uses distinct hydrophobic surfaces to recognize type 1 (PPYPXYYY) vs type 2 (PXPGF) motifs.\",\n      \"method\": \"X-ray crystallography, single amino acid substitution mutagenesis, pulldown binding assays\",\n      \"journal\": \"International Journal of Molecular Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus systematic mutagenesis defining mechanistically distinct binding pockets, single lab\",\n      \"pmids\": [\"25667979\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Ca2+-dependent ALG-2 interaction with ALIX relieves the default intramolecular autoinhibitory interaction of ALIX, promoting CHMP4-dependent ALIX membrane association; EGFR activation increases ALG-2-ALIX interaction, and this is required for ALIX-mediated MVB sorting of activated EGFR; ALG-2 activation of ALIX does not affect cytokinetic abscission or EIAV budding.\",\n      \"method\": \"Pulldown assays, membrane fractionation, EGFR MVB sorting assay, co-transfection with dominant-negative ALIX mutants, EIAV budding assay\",\n      \"journal\": \"Cell Discovery\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple functional assays distinguishing pathway-specific ALG-2 effects, direct mechanism of ALIX activation demonstrated\",\n      \"pmids\": [\"27462417\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ALG-2 promotes ER exit site (ERES) localization and Ca2+-dependent polymerization of TFG (Trk-fused gene protein); ALG-2 interacts only with the ALG-2 homodimer (not ALG-2/peflin heterodimer); TFG deletion of the ALG-2-binding motif shortens TFG half-life at ERES; ALG-2 overexpression accumulates TFG at ERES.\",\n      \"method\": \"Co-immunoprecipitation, immunostaining, live-cell time-lapse imaging (thapsigargin-induced Ca2+ rise), in vitro cross-linking polymerization assay, motif deletion analysis\",\n      \"journal\": \"The FEBS Journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — live imaging plus in vitro polymerization assay plus deletion mutagenesis, multiple methods in one study\",\n      \"pmids\": [\"27813252\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ALG-2 interacts Ca2+-dependently with MISSL (MAPK1-interacting and spindle-stabilizing-like protein); MISSL and ALG-2 co-relocalize to puncta upon Ca2+ rise; knockdown of either MISSL or ALG-2 reduces secreted alkaline phosphatase secretion and delays ER-to-Golgi transport of procollagen I; MISSL and ALG-2 also interact with MAP1B, and MAP1B knockdown reverses reduced secretion caused by MISSL/ALG-2 depletion.\",\n      \"method\": \"Co-immunoprecipitation, live-cell imaging, siRNA knockdown with secretion assay (SEAP), procollagen I transport assay, epistasis by double knockdown\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP plus functional secretion assay plus genetic epistasis (double knockdown), multiple orthogonal methods\",\n      \"pmids\": [\"28864773\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ALG-2 interacts with HEBP2 (heme-binding protein 2); co-expression increases the cytoplasmic pool of ALG-2 and alters HEBP2 distribution; the ALG-2/HEBP2 complex affects mitotic spindle orientation/positioning and microtubule dynamics in cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, subcellular localization imaging, mitotic spindle analysis, microtubule dynamics assay\",\n      \"journal\": \"Journal of Cellular Physiology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP plus imaging, limited mechanistic detail in abstract, single lab\",\n      \"pmids\": [\"28004381\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"ALG-2 participates in plasma membrane repair; ALG-2 knockout DT-40 cells are more sensitive to electroporation; reintroduction of ALG-2 rescues this sensitivity; wild-type but not Ca2+-binding-deficient ALG-2 partially protects HeLa cells from digitonin-induced death; a peptide with the ALG-2 binding sequence of ALIX inhibits this protective function.\",\n      \"method\": \"PDCD6 gene disruption in DT-40 cells, electroporation survival assay, overexpression rescue, digitonin treatment, ALIX peptide competition\",\n      \"journal\": \"PLoS One\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — gene knockout plus rescue plus peptide competition, Ca2+-binding requirement confirmed, multiple lines of evidence\",\n      \"pmids\": [\"30240438\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"MAP1B interacts Ca2+-dependently with ALG-2 through a region lacking canonical ABM-1/ABM-2 motifs; MAP1B binding selectively competes with ABM-2-containing proteins (e.g., Sec31A) for ALG-2; MAP1B knockout cells show increased co-localization of ALG-2 with Sec31A; overexpression of wild-type MAP1B disperses ALG-2 and Sec31A distributions.\",\n      \"method\": \"Co-immunoprecipitation, point mutagenesis of MAP1B, MAP1B knockout cells, immunofluorescence co-localization\",\n      \"journal\": \"Biochemical and Biophysical Research Communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus KO cells plus imaging, single lab, multiple methods\",\n      \"pmids\": [\"29432744\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ALG-2 directly interacts with Rpn3 (a component of the 26S proteasome) and regulates proteasome activity upon Ca2+ elevation following T cell activation; this modulates MCL1 stability and accelerates T cell apoptosis (contraction); ALG-2 thus couples T cell activation to the subsequent apoptotic contraction phase.\",\n      \"method\": \"Co-immunoprecipitation (ALG-2-Rpn3), proteasome activity assay, MCL1 stability assay, T cell activation and apoptosis assays\",\n      \"journal\": \"Cell Death & Disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus functional assays for proteasome activity and MCL1 stability, single lab\",\n      \"pmids\": [\"31919392\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"PDCD6 interacts with c-Raf via Co-IP, leading to activation of the c-Raf/MEK/ERK MAPK pathway and upregulation of MYC and JUN, promoting colorectal cancer cell proliferation.\",\n      \"method\": \"Co-immunoprecipitation, mass spectrometry, RNA-seq, Western blot of MAPK pathway components, in vitro and in vivo proliferation assays\",\n      \"journal\": \"Journal of Experimental & Clinical Cancer Research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP plus downstream signaling, limited mechanistic dissection of direct interaction, single lab\",\n      \"pmids\": [\"32746883\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ALG-2 and peflin together constitute a hetero-bifunctional COPII regulator; at steady-state Ca2+, ALG-2/peflin heterocomplexes bound to ERES confer a buffered secretion rate, while peflin-lacking ALG-2 complexes stimulate secretion; upon Ca2+ signaling, ALG-2-dependent effects on secretion can either increase or decrease ER export depending on signaling intensity; mechanistically, depression of secretion involves decreased COPII outer shell and increased peflin at ERES, while enhancement involves increased COPII outer shell and decreased peflin.\",\n      \"method\": \"ER-to-Golgi transport assays in NRK and PC12 cells, Ca2+ mobilization by ATP, COPII protein fractionation, peflin/ALG-2 siRNA knockdowns, secretion of physiological cargoes (collagen I, SEAP)\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — functional transport assay in two cell types, bidirectional Ca2+ effect, mechanistic dissection of heterodimer vs homodimer roles, multiple methods\",\n      \"pmids\": [\"34762908\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CDIP1 (cell death-inducing p53 target 1) interacts with ALG-2 Ca2+-dependently; ALG-2 promotes CDIP1 association with ESCRT-I (preferentially VPS37B/C-containing); co-expression of ALG-2 and ESCRT-I enhances CDIP1-induced caspase-3/7-mediated cell death; CDIP1 also binds VAPA/B via an FFAT-like motif.\",\n      \"method\": \"Co-immunoprecipitation, Ca2+-dependent pulldown, overexpression with caspase activity assay, domain deletion analysis\",\n      \"journal\": \"International Journal of Molecular Sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus functional cell death assay, single lab, multiple methods\",\n      \"pmids\": [\"33503978\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"MAT2A interacts with PDCD6 and, upon AMPK activation, facilitates methylation of PDCD6 at K90, which increases PDCD6 protein stability; K90R mutation increases apoptosis and suppresses cervical cancer cell growth under glucose deprivation.\",\n      \"method\": \"Co-immunoprecipitation, immunoblotting, mass spectrometry, AMPK pathway inhibitors, K90R point mutation, cell viability and apoptosis assays\",\n      \"journal\": \"Cell Death Discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus MS identification of methylation site plus mutagenesis with functional readout, single lab\",\n      \"pmids\": [\"35396512\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ALG-2 binds directly to acidic membranes in a Ca2+-dependent manner via electrostatic and hydrophobic interactions; charge-reversed mutants disrupt membrane recruitment; membrane binding is required for ERES localization but ESCRT-I binding can rescue membrane-binding-defective ALG-2 at lysosomes; Ca2+-dependent membrane binding and protein binding act together in cellular ALG-2 functions.\",\n      \"method\": \"Giant unilamellar vesicle (GUV) binding experiments, molecular dynamics simulations, charge-reversed mutagenesis, fluorescence imaging in cells (thapsigargin and lysosomal Ca2+ release), in vitro reconstitution with ESCRT-I\",\n      \"journal\": \"Proceedings of the National Academy of Sciences USA\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution plus MD simulations plus mutagenesis plus cellular imaging, multiple orthogonal methods\",\n      \"pmids\": [\"38386713\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ALG-2, upon lysosomal Ca2+ release (e.g., GPN-induced osmotic stress or TRPML1 activation), redistributes onto lysosomes and recruits ESCRT proteins, enhancing lysosomal membrane resilience to osmotic rupture; the ALG-2(ΔGF122) splice variant defective in ESCRT binding does not confer this protection; chelating cytoplasmic Ca2+ sensitizes lysosomes to rupture.\",\n      \"method\": \"Lysosomal leakage/rupture assays (sensitive fluorescent reporters), Ca2+ chelation (BAPTA), GPN and TRPML1 agonist treatments, ALG-2 and ΔGF122 overexpression, ESCRT recruitment imaging\",\n      \"journal\": \"Proceedings of the National Academy of Sciences USA\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple functional assays of lysosomal integrity, isoform control, Ca2+ manipulation, ESCRT recruitment, single lab but rigorous multi-method design\",\n      \"pmids\": [\"38781205\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PDCD6 interacts with LDHA and downregulates lactate metabolism; PDCD6 deficiency increases LDHA activity and lactate production, leading to RUBCN lactylation at K33, which promotes RUBCN interaction with VPS34, LAP (LC3-associated phagocytosis) formation, and bactericidal activity.\",\n      \"method\": \"Co-immunoprecipitation (PDCD6-LDHA), genetic knockout in mice and macrophages, LDHA pharmacological inhibition, lactate measurement, RUBCN lactylation site identification, LAP assays, bacterial killing assays\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP plus KO mouse model plus biochemical pathway dissection including PTM identification, multiple methods in one study\",\n      \"pmids\": [\"39578445\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"PDCD6 interacts with death-associated protein kinase 1 (DAPk1); co-transfection of PDCD6 and DAPk1 additively accelerates apoptosis via a caspase-3 dependent pathway.\",\n      \"method\": \"Yeast two-hybrid screening of human ovary cDNA library, co-transfection apoptosis assay with caspase-3 readout\",\n      \"journal\": \"Biotechnology Letters\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — yeast two-hybrid only for interaction, single cell line, single lab\",\n      \"pmids\": [\"16132846\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"ALG-2 knockdown in HeLa cells causes G2/M cell cycle arrest and increased early apoptosis/cell death; pan-caspase inhibitor zVAD-fmk attenuates the increase in dead cells, indicating ALG-2 has an anti-apoptotic function in HeLa cells by facilitating G2/M checkpoint passage.\",\n      \"method\": \"siRNA knockdown, cell cycle analysis by flow cytometry, cell death quantification, caspase inhibitor (zVAD-fmk) rescue\",\n      \"journal\": \"Biochemical and Biophysical Research Communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA loss-of-function with cell cycle and death readouts plus pharmacological rescue, single lab\",\n      \"pmids\": [\"19013425\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ALG-2 interacts Ca2+-dependently with SARAF (a negative regulator of store-operated Ca2+ entry); ALG-2 overexpression interferes with NEDD4-family E3 ligase-mediated ubiquitination of SARAF at PPXY motifs proximal to the ALG-2 binding site, stabilizing SARAF; ALG-2 dimer promotes Ca2+-dependent SARAF CytD-to-CytD interactions.\",\n      \"method\": \"Semi-quantitative in vitro binding assay, pulldown with ubiquitination assay, half-life analysis, Strep-tag pulldown, Lys-to-Arg substitution mutants\",\n      \"journal\": \"International Journal of Molecular Sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — binding assay plus functional ubiquitination/stability analysis with mutagenesis, single lab\",\n      \"pmids\": [\"32878247\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PDCD6 interacts with the intracellular domain of cell adhesion molecule CHL1 in a Ca2+-dependent manner (Ca2+ chelation with BAPTA-AM abolishes association); a cell-penetrating CHL1-ICD peptide inhibits both the CHL1-PDCD6 association and PDCD6/CHL1-triggered neuronal survival.\",\n      \"method\": \"Co-immunoprecipitation, GST pull-down, proximity ligation assay in mouse brain tissue and cultured neurons, BAPTA-AM Ca2+ chelation, cell-penetrating peptide inhibition\",\n      \"journal\": \"FASEB BioAdvances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus PLA in tissue plus functional peptide inhibition, single lab\",\n      \"pmids\": [\"35024572\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"PATL1 (P-body component Pat1b) is a novel ALG-2-interacting protein; endogenous PATL1 and ALG-2 co-immunoprecipitate; a subset of ALG-2 co-localizes with PATL1 and the P-body marker DCP1A, identifying ALG-2 as having a potential role at P-bodies.\",\n      \"method\": \"In silico ABM screening, Far-Western blot, co-immunoprecipitation with endogenous proteins, immunofluorescence co-localization\",\n      \"journal\": \"Journal of Biochemistry\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP plus co-localization only, no functional consequence established, single lab\",\n      \"pmids\": [\"22437941\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PDCD6/ALG-2 is a penta-EF-hand Ca2+-binding protein that acts as a multifunctional Ca2+-dependent adaptor: upon Ca2+ binding, an arginine-switch mechanism (Arg125, driven by Ca2+ at EF3) opens hydrophobic pockets that recognize Pro-rich motifs (type 1: PPYPXYYY in ALIX/TSG101; type 2: PXPGF in Sec31A) in diverse partners, enabling ALG-2 homodimers to bridge ALIX and ESCRT-I on endosomes to promote MVB sorting and lysosomal membrane repair, to stabilize the COPII outer coat protein Sec31A at ER exit sites to regulate ER-to-Golgi vesicular transport, to polymerize TFG and recruit MISSL/MAP1B at ERES, to shuttle to the nucleus with RBM22 and regulate alternative splicing of IP3R1 pre-mRNA via CHERP, to interact with the 26S proteasome subunit Rpn3 and modulate MCL1 stability during T cell contraction, to downregulate lactate metabolism via LDHA thereby controlling RUBCN lactylation and LC3-associated phagocytosis, and to participate in plasma membrane repair—all in a Ca2+-dependent and dimerization-dependent fashion, explaining its original identification as a required mediator of Ca2+-regulated apoptosis acting downstream of caspases.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PDCD6/ALG-2 is a penta-EF-hand Ca2+-binding protein that functions as a Ca2+-sensing molecular adaptor coupling intracellular Ca2+ transients to membrane traffic, membrane repair, and cell-fate decisions [#0, #6]. Ca2+ binding at its high-affinity EF-hands triggers a conformational change that exposes a hydrophobic surface [#2, #4], and crystallographic work resolved this as an arginine-switch in which Ca2+ loading at EF3 repositions Arg125 to open hydrophobic pockets that capture Pro-rich peptide motifs in partner proteins [#19]; distinct pockets recognize type 1 (PPYP, as in ALIX/TSG101) versus type 2 (PXPGF, as in Sec31A) motifs, and the alternatively spliced ALG-2(ΔGF122) isoform reshapes these pockets to alter target selectivity [#21, #25, #31]. Functional ALG-2 acts as a homodimer (mediated by the fifth EF-hand) and additionally binds acidic membranes Ca2+-dependently, with membrane and peptide engagement acting together for recruitment [#4, #43]. As an adaptor it bridges ALIX and ESCRT-I, relieves ALIX autoinhibition to promote CHMP4-dependent membrane association, and thereby drives MVB sorting of activated EGFR and recruits ESCRT to reinforce lysosomal membranes against rupture [#22, #32, #44]. At ER exit sites ALG-2 binds and stabilizes the COPII outer-coat protein Sec31A and, together with its paralog peflin, constitutes a hetero-bifunctional Ca2+-tunable regulator of ER-to-Golgi secretion, also polymerizing TFG and recruiting MISSL/MAP1B [#14, #30, #33, #34, #40]. In the nucleus ALG-2 partners with CHERP to regulate alternative splicing of IP3R1 pre-mRNA [#28]. ALG-2 was originally identified as a required mediator of Ca2+-regulated apoptosis acting downstream of caspase activation [#0, #1], and it further couples Ca2+ signals to protein turnover and metabolism by engaging the proteasome subunit Rpn3 to control MCL1 during T-cell contraction and by restraining LDHA-driven lactate production that governs RUBCN lactylation and LC3-associated phagocytosis [#38, #45].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Established that ALG-2 is a required component of Ca2+-regulated programmed cell death, defining the gene's founding biological role.\",\n      \"evidence\": \"Death-trap functional selection and antisense depletion in T cell hybridoma cells across TCR, Fas, and glucocorticoid death stimuli\",\n      \"pmids\": [\"8560270\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism by which ALG-2 enables death not defined\", \"No direct partners identified at this stage\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Placed ALG-2 at a post-caspase step in apoptosis, distinguishing its function from upstream protease activation.\",\n      \"evidence\": \"Caspase activity and PARP cleavage assays in ALG-2-depleted clones showing normal cleavage yet death protection\",\n      \"pmids\": [\"9164928\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The downstream effectors ALG-2 acts through were not identified\", \"Did not link the apoptotic role to a biochemical activity\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Defined the Ca2+-dependent conformational logic and dimerization of ALG-2, showing Ca2+ binding exposes a hydrophobic surface and EF5 drives homodimerization.\",\n      \"evidence\": \"TNS fluorescence, gel filtration, cross-linking, CD and EF-hand mutagenesis on recombinant protein\",\n      \"pmids\": [\"9832622\", \"10360947\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No physiological binding partner mapped to the exposed surface yet\", \"Cellular consequence of the conformational change unresolved\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Identified ALIX/AIP1 as the first strictly Ca2+-dependent ALG-2 partner cooperating in cell death, linking the conformational switch to a functional interaction.\",\n      \"evidence\": \"Yeast two-hybrid, reciprocal Co-IP, co-localization, and overexpression rescue, confirmed by a parallel study\",\n      \"pmids\": [\"9880530\", \"10200558\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the interaction not yet resolved\", \"Direct molecular role of the ALG-2-ALIX complex undefined\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Generalized ALG-2 target recognition to a Pro/Gly/Tyr-rich binding mode by reconstituting direct nanomolar Ca2+-dependent binding to annexin VII/XI N-termini.\",\n      \"evidence\": \"SPR kinetics, GST pull-down, and biotin-ALG-2 overlay with recombinant annexins\",\n      \"pmids\": [\"11883939\", \"12445460\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional outcome of annexin binding in cells not established\", \"Did not yet define the precise peptide motif consensus\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Resolved the first Ca2+-loaded ALG-2 crystal structure, showing a hinge rotation at EF3 that exposes a peptide-accepting cleft at the dimer interface.\",\n      \"evidence\": \"X-ray crystallography at 2.3 Å of Ca2+-bound ALG-2\",\n      \"pmids\": [\"11525164\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not capture a bound target peptide\", \"Residue-level switch mechanism not yet defined\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Extended ALG-2 into the ESCRT machinery by showing direct Ca2+-dependent binding to TSG101 and Ca2+-dependent endosomal recruitment.\",\n      \"evidence\": \"GST pull-down, yeast two-hybrid, overlay, and immunofluorescence with BAPTA-AM chelation in cells\",\n      \"pmids\": [\"16004603\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ALG-2 bridges ALIX and ESCRT-I not yet tested\", \"Functional consequence for endosomal sorting undefined\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Connected ALG-2 to the early secretory pathway by demonstrating Ca2+-dependent recruitment to ER exit sites via Sec31A and reciprocal stabilization of Sec31A.\",\n      \"evidence\": \"Ca2+-dependent pull-down, overlay, RNAi, confocal imaging and pharmacological Ca2+ manipulation, independently replicated\",\n      \"pmids\": [\"16957052\", \"17196169\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Effect on actual cargo transport rates not yet measured\", \"Distinct binding mode versus ALIX/TSG101 not yet resolved\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Placed ALG-2 in the nucleus through RBM22-mediated import, hinting at a role beyond membrane traffic.\",\n      \"evidence\": \"Yeast two-hybrid and confocal co-localization in NIH 3T3 cells and zebrafish embryos\",\n      \"pmids\": [\"17045351\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct binding reconstitution\", \"Nuclear function not established at this stage\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Defined the residue-level arginine-switch mechanism, showing Ca2+/EF3 repositions Arg125 to open the primary hydrophobic pocket that captures the ALIX PPYP motif.\",\n      \"evidence\": \"Multiple crystal structures including the ALG-2/Alix peptide complex plus mutagenesis-validated binding\",\n      \"pmids\": [\"18940611\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve type 2 motif recognition\", \"Membrane contribution to recruitment not addressed\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Resolved a second, distinct ALG-2 binding pocket and motif class, explaining how the protein discriminates type 1 (PPYP) from type 2 (PXPGF) ligands.\",\n      \"evidence\": \"SPR, overlay, GST pull-down and mutagenesis on PLSCR3 ABS-1/ABS-2 sites\",\n      \"pmids\": [\"18256029\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cellular role of PLSCR3 binding undefined\", \"Structural detail of the second pocket awaited a co-crystal\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Established ALG-2 as a Ca2+-dependent dimeric adaptor that physically bridges ALIX and TSG101, unifying its ESCRT-associated functions.\",\n      \"evidence\": \"Knockdown plus recombinant add-back ternary-complex reconstitution with isoform and dimerization mutant controls\",\n      \"pmids\": [\"19520058\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Membrane context of bridging not addressed\", \"Downstream sorting cargo not yet defined in this study\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Resolved the structural basis of isoform-selective target recognition, showing ΔGF122 repositions Arg125 to block Pocket 1 and differentially affect partner binding.\",\n      \"evidence\": \"Crystal structure of ALG-2(ΔGF122) plus systematic mutant binding assays against Alix/TSG101/annexin A11\",\n      \"pmids\": [\"20691033\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological role of isoform switching not established\", \"Did not capture the type 2 pocket structurally\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Demonstrated that ALG-2/Sec31A engagement functionally controls ER-to-Golgi transport and cargo receptor stability, not merely Sec31A localization.\",\n      \"evidence\": \"Intact-cell transport assays, EM, Ca2+ depletion, and binding-domain disruption with p24/p115 analysis\",\n      \"pmids\": [\"25006245\", \"24069399\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direction of regulation (stimulatory vs inhibitory) not fully reconciled\", \"Role of peflin not yet integrated\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Assigned ALG-2 a nuclear function in alternative splicing, linking Ca2+ signaling to IP3R1 pre-mRNA processing via CHERP.\",\n      \"evidence\": \"Co-IP, live imaging, RNA-IP, and siRNA knockdown with RT-PCR splicing analysis\",\n      \"pmids\": [\"24078636\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Breadth of ALG-2-dependent splicing targets unknown\", \"Mechanism of ALG-2 within the spliceosome undefined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Resolved the third hydrophobic pocket recognizing the Sec31A type 2 PXPGF motif and validated pocket-specific mutations separating Sec31A from Alix binding.\",\n      \"evidence\": \"Crystallography of the ALG-2/Sec31A peptide complex plus single-residue substitution binding assays\",\n      \"pmids\": [\"25667979\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo significance of pocket separation not tested\", \"Did not address membrane-binding contribution\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Established that ALG-2 functions selectively as a homodimer at ERES to polymerize TFG and recruit MISSL/MAP1B, expanding its ERES regulatory repertoire.\",\n      \"evidence\": \"Co-IP, live imaging, in vitro polymerization assays, and double-knockdown epistasis with secretion readouts\",\n      \"pmids\": [\"27813252\", \"28864773\", \"29432744\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How homodimer vs heterodimer selection is regulated in cells unclear\", \"Competition between MAP1B and Sec31A binding not fully mapped physiologically\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Defined the molecular consequence of ALG-2-ALIX binding as relief of ALIX autoinhibition driving pathway-specific MVB sorting of activated EGFR.\",\n      \"evidence\": \"Pull-down, membrane fractionation, EGFR MVB sorting, and dominant-negative ALIX assays distinguishing pathway specificity\",\n      \"pmids\": [\"27462417\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why ALG-2 affects MVB sorting but not abscission/budding not fully explained\", \"In vivo relevance not established\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Linked ALG-2 to regulated protein turnover by showing direct Rpn3/proteasome engagement that controls MCL1 stability during T-cell contraction.\",\n      \"evidence\": \"Co-IP, proteasome activity assays, and MCL1 stability with T-cell activation/apoptosis readouts\",\n      \"pmids\": [\"31919392\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether Rpn3 binding is direct via the canonical pockets unresolved\", \"Reciprocal validation of proteasome regulation limited to single lab\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Integrated ALG-2 with peflin into a hetero-bifunctional COPII regulator that bidirectionally tunes ER export according to Ca2+ signal intensity.\",\n      \"evidence\": \"ER-to-Golgi transport assays in two cell types with COPII fractionation and ALG-2/peflin knockdowns\",\n      \"pmids\": [\"34762908\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative threshold setting between buffering and stimulation undefined\", \"Structural basis of heterodimer COPII effect not resolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstrated that ALG-2 directly engages acidic membranes Ca2+-dependently and that membrane binding and ESCRT-I binding act together for compartment-specific recruitment.\",\n      \"evidence\": \"GUV binding, molecular dynamics, charge-reversal mutagenesis, and cellular imaging with in vitro ESCRT-I reconstitution\",\n      \"pmids\": [\"38386713\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of membrane vs peptide binding across partners not quantified\", \"Lipid specificity determinants only partly defined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Assigned ALG-2 a protective role in lysosomal membrane repair via Ca2+-triggered ESCRT recruitment, with the ESCRT-binding-defective ΔGF122 isoform unable to confer protection.\",\n      \"evidence\": \"Lysosomal rupture reporters, Ca2+ chelation, GPN/TRPML1 agonists, and isoform-controlled ESCRT recruitment imaging\",\n      \"pmids\": [\"38781205\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo physiological setting of lysosomal repair not tested\", \"Crosstalk with plasma-membrane repair function not delineated\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Connected PDCD6 to immunometabolism by showing it restrains LDHA-driven lactate production, controlling RUBCN lactylation and LC3-associated phagocytosis.\",\n      \"evidence\": \"Co-IP, knockout mice/macrophages, LDHA inhibition, lactylation site mapping, and bactericidal LAP assays\",\n      \"pmids\": [\"39578445\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PDCD6-LDHA binding uses the canonical Ca2+/peptide mechanism unclear\", \"Ca2+ dependence of this metabolic axis not addressed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the diverse ALG-2 functions—secretory traffic, ESCRT-dependent membrane repair, nuclear splicing, proteasome and metabolic regulation—are coordinated and prioritized by distinct Ca2+ signal patterns within a single cell remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No integrated model of partner selection under physiological Ca2+ dynamics\", \"In vivo phenotypes tying the molecular activities to organismal physiology largely lacking\", \"Mechanistic basis for several Co-IP-only partners (c-Raf, VEGFR-2, DAPk1, PATL1) not established\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [22, 29, 32]},\n      {\"term_id\": \"GO:0140299\", \"supporting_discovery_ids\": [2, 6, 15, 19]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [43]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [14, 33, 48]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [3, 7]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [13, 32]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [14, 30, 33]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [43, 44]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [7, 16, 28]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [36, 43]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [14, 30, 32, 40]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [0, 1, 38]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [36, 44]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [28]}\n    ],\n    \"complexes\": [\"ESCRT-I/ALIX/ALG-2 ternary complex\", \"ALG-2 homodimer\", \"ALG-2/peflin heterodimer\", \"COPII outer coat (Sec13/31A)\"],\n    \"partners\": [\"PDCD6IP/ALIX\", \"TSG101\", \"SEC31A\", \"PEF1\", \"ANXA11\", \"TFG\", \"CHERP\", \"PSMD3\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}