{"gene":"TP53BP2","run_date":"2026-06-10T10:51:55","timeline":{"discoveries":[{"year":1996,"finding":"Crystal structure of the p53 core domain bound to 53BP2 (ASPP2 C-terminal fragment) revealed that the SH3 domain of 53BP2 binds the L3 loop of p53 in a manner distinct from canonical SH3-polyproline complexes, and an ankyrin repeat binds the L2 loop of p53; the binding site overlaps the p53 DNA-binding surface and the six most frequent cancer-associated p53 mutations disrupt 53BP2 binding in vitro.","method":"X-ray crystallography (crystal structure of p53 core domain–53BP2 complex); in vitro binding assays with cancer-associated p53 mutants","journal":"Science","confidence":"High","confidence_rationale":"Tier 1 / Strong — atomic-resolution crystal structure with functional in vitro validation; foundational structural paper replicated by subsequent studies","pmids":["8875926"],"is_preprint":false},{"year":1996,"finding":"53BP2/ASPP2 interacts with Bcl-2 via its ankyrin repeats and SH3 domain (same domains that mediate p53 binding); Bcl-2 and p53 compete for binding to 53BP2 in vitro; overexpressed 53BP2 partially colocalizes with Bcl-2 in the cytoplasm and increases the proportion of cells at G2/M.","method":"Yeast two-hybrid screen; in vitro GST pull-down with bacterially expressed proteins; competition binding assays; immunofluorescence colocalization; cell cycle analysis by flow cytometry","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal yeast two-hybrid plus in vitro reconstitution with competition experiments, replicated in subsequent work","pmids":["8668206"],"is_preprint":false},{"year":1998,"finding":"53BP2 localizes exclusively to the cytoplasm (not altered by co-expression of wild-type p53); despite this, both 53BP1 and 53BP2 enhance p53-mediated transcriptional activation in cell-based reporter assays, suggesting they function in signal transduction pathways to promote p53 activity without forming a concurrent DNA-binding complex with p53.","method":"Immunofluorescence subcellular localization; p53-dependent transcriptional reporter assays; Western blot for p53 target protein induction","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct localization experiment and reporter assay from single lab with two orthogonal methods","pmids":["9748285"],"is_preprint":false},{"year":1999,"finding":"NF-κB p65 (RelA) subunit directly binds 53BP2/ASPP2 via yeast two-hybrid and in vitro pull-down; co-expression of p65 significantly inhibits 53BP2-induced apoptosis; full-length GFP-53BP2 localizes to perinuclear cytoplasmic puncta and induces apoptosis, whereas N-terminal or C-terminal fragments alone do not.","method":"Yeast two-hybrid; in vitro pull-down assay; mammalian two-hybrid assay; GFP fusion subcellular localization; apoptosis assay by transfection","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid confirmed by independent in vitro pull-down and mammalian two-hybrid; single lab","pmids":["10498867"],"is_preprint":false},{"year":2000,"finding":"Endogenous 53BP2/ASPP2 protein levels increase following UV-irradiation-induced DNA damage in a p53-independent manner; conversely, wild-type (but not mutant) p53 suppresses 53BP2 steady-state protein levels; conditional expression of 53BP2 lowers the apoptotic threshold after UV irradiation, and antisense attenuation of 53BP2 induction enhances clonogenic survival.","method":"Western blot of endogenous protein in cell lines with defined p53 genotypes; tetracycline-regulated p53 expression system; ecdysone-regulated 53BP2 stable cell lines; antisense oligonucleotide knockdown; clonogenic survival assay; apoptosis assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal genetic approaches (KO cell lines, inducible OE, antisense KD) with defined phenotypic readouts; single lab but rigorous","pmids":["11027272"],"is_preprint":false},{"year":2002,"finding":"ASPP1 and ASPP2 selectively stimulate the apoptotic transcriptional function of p53 (activating Bax, PIG3, PUMA promoters) but not cell-cycle arrest targets (mdm2, p21); they also bind p63 and p73 in vitro and in vivo and stimulate their apoptotic function; RNAi depletion of endogenous p63/p73 demonstrated that the p53-independent apoptotic activity of ASPP1/ASPP2 is mainly mediated by p63/p73.","method":"Co-immunoprecipitation (in vivo); in vitro binding; luciferase reporter assays on apoptotic vs. cell-cycle promoters; RNA interference knockdown of p63/p73; apoptosis assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP plus RNAi epistasis plus promoter-selective reporter assays; multiple orthogonal methods in one rigorous study","pmids":["14729977"],"is_preprint":false},{"year":2003,"finding":"ASPP2 specifically interacts with APP-BP1 (the NEDD8-activating enzyme subunit) in non-transfected cells through the N-terminal domain ASPP2(332–483); ASPP2 inhibits APP-BP1-mediated NEDD8 conjugation to Cullin-1, reduces APP-BP1-induced cell proliferation, and blocks APP-BP1-triggered apoptosis in primary neurons; ASPP2 also activates NF-κB transcriptional activity.","method":"Co-immunoprecipitation from non-transfected cells; domain mapping; NEDD8 conjugation assay (in vitro/cellular); cell proliferation and neuronal apoptosis assays","journal":"Journal of neurochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — endogenous Co-IP plus functional neddylation assay; single lab, two orthogonal methods","pmids":["12694406"],"is_preprint":false},{"year":2004,"finding":"Hepatitis C virus core protein interacts with 53BP2/ASPP2 in a yeast two-hybrid assay; the core protein competes with p53 for binding to ASPP2 in vitro and, when co-expressed, inhibits ASPP2-enhanced p53-mediated apoptosis without affecting p53 transcriptional activity on Bax or p21 promoters.","method":"Yeast two-hybrid; in vitro competition binding assay; apoptosis assay; reporter assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid confirmed by in vitro competition and cellular apoptosis assay; single lab","pmids":["14985081"],"is_preprint":false},{"year":2004,"finding":"The TP53BP2 gene encodes two protein isoforms—53BP2S (short, 1005 aa) and 53BP2L/ASPP2 (long, 1128 aa, with an additional N-terminal 123 aa encoded by exon 3)—generated by alternative splicing.","method":"RT-PCR and genomic cloning of TP53BP2 transcripts; sequencing","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Strong — direct transcript sequencing from multiple cell lines and tissues; independently confirmed in subsequent studies","pmids":["14766226"],"is_preprint":false},{"year":2005,"finding":"ASPP2/53BP2L is a transcriptional target of E2F: E2F-1, -2, and -3 bind the endogenous ASPP2 promoter (demonstrated by ChIP), and ectopic E2F-1 increases endogenous ASPP2 mRNA and protein; ASPP2 expression is maximal in early S-phase.","method":"Chromatin immunoprecipitation (ChIP); luciferase promoter-reporter assays with E2F binding site mutants; Western blot and RT-PCR of endogenous protein after E2F-1 induction; cell-cycle-staged expression analysis","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 2 / Strong — ChIP of endogenous promoter plus mutational promoter analysis plus endogenous protein induction; two independent papers (PMIDs 15592436 and 15731768) replicate the finding","pmids":["15592436","15731768"],"is_preprint":false},{"year":2005,"finding":"ASPP2/53BP2L protein is subject to proteasomal degradation; proteasomal inhibitors (including bortezomib) and anthracycline-based chemotherapy increase ASPP2 protein but not mRNA levels by stabilizing the protein; the central region of ASPP2 is ubiquitinated; siRNA knockdown of ASPP2 attenuates bortezomib-induced apoptosis, particularly in p53 wild-type cells.","method":"Proteasome inhibitor treatment; cycloheximide chase (protein half-life); ubiquitination assay; siRNA knockdown; apoptosis assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — protein stability assay combined with ubiquitination detection and functional knockdown; single lab","pmids":["16091363"],"is_preprint":false},{"year":2005,"finding":"53BP2 binding to p53 and DNA binding are mutually exclusive (no ternary complex detected for GADD45, p21, Bax, or PIG3 response elements); multiple oncogenic p53 mutations (R181E, G245S, R249S, R273H) differentially affect DNA and 53BP2 binding.","method":"Biophysical binding assays (fluorescence anisotropy, isothermal titration calorimetry, SPR); competition experiments with DNA response elements and recombinant proteins; p53 mutant panel","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — quantitative in vitro biophysical assays with mutagenesis panel; single lab but multiple orthogonal biophysical methods","pmids":["16887812"],"is_preprint":false},{"year":2005,"finding":"Mdm2 and MdmX prevent ASPP1/ASPP2 from stimulating the apoptotic function of p53 by binding and inhibiting the transcriptional activity of p53, without targeting p53 for degradation; both the DNA-binding and transactivation functions of p53 are required for ASPP1/ASPP2 stimulation.","method":"p53/mdm2 mutant panel; transcriptional reporter assays; co-immunoprecipitation; Western blot for p53 stability","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic mutant dissection plus reporter assays; single lab with two orthogonal approaches","pmids":["15782125"],"is_preprint":false},{"year":2005,"finding":"53BP2/ASPP2 induces apoptosis via the mitochondrial death pathway: it localizes to mitochondria, causes depression of mitochondrial transmembrane potential (ΔΨm), and activates caspase-9 leading to PARP cleavage.","method":"Subcellular fractionation; mitochondrial membrane potential assay (JC-1); caspase-9 activity assay; annexin V staining; PARP cleavage Western blot","journal":"Genes to cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — subcellular fractionation localization plus functional mitochondrial assays; single lab","pmids":["15743414"],"is_preprint":false},{"year":2008,"finding":"ASPP2 C-terminal ankyrin repeats and SH3 domain (ASPP2 Ank-SH3) mediate interaction with Bcl-2 family members (Bcl-2, Bcl-xL, Bcl-w) at two sites: the conserved BH4 motif and a proapoptotic regulator-binding site; within Bcl-2, binding to the BH4 domain is tightest; based on docking analysis ASPP2 is proposed to inhibit Bcl-2 function by occupying functional sites.","method":"Peptide array screening; surface plasmon resonance (SPR); isothermal titration calorimetry (ITC); computational docking; sequence alignment and peptide mutagenesis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Moderate — quantitative biophysical binding assays (SPR + ITC) plus peptide array plus structural modeling; multiple orthogonal methods in single rigorous study","pmids":["18719108"],"is_preprint":false},{"year":2008,"finding":"The C-termini of ASPP1 and ASPP2 directly bind the DNA-binding domains of p53, p63, and p73 with dissociation constants in the low micromolar range in a 1:1 stoichiometry; tri-complex formation between ASPPs, p53 family members, and PUMA/Bax DNA is mutually exclusive; uniquely, ASPP2 (but not ASPP1) forms a complex with PUMA and displaces p53 and p73.","method":"Surface plasmon resonance; isothermal titration calorimetry; EMSA (electrophoretic mobility shift assay); structure-based homology modelling","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — quantitative SPR and ITC plus EMSA; multiple orthogonal biochemical methods in one study","pmids":["18676979"],"is_preprint":false},{"year":2008,"finding":"The proline-rich domain of ASPP2 is natively unfolded and forms an intramolecular autoinhibitory interaction with its own Ank-SH3 domains, competing with intermolecular partner binding; ASPP2 Ank-SH3 (not the Pro domain) mediates interactions with partner-derived peptides; the presence of the Pro domain inhibits interactions mediated by Ank-SH3.","method":"CD spectroscopy; NMR; size exclusion chromatography; fluorescence anisotropy; peptide array screening; GST pull-down","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR and CD structural characterization plus multiple biochemical binding assays; multiple orthogonal methods in one study","pmids":["18448430"],"is_preprint":false},{"year":2009,"finding":"ASPP2 binds Par-3 and controls its apical/junctional localization in neural progenitors; junctional localization of ASPP2 and Par-3 is interdependent; loss of ASPP2 in vivo disrupts tight/adherens junctions, impairs interkinetic nuclear migration, and causes neuroblastic rosette formation resembling primitive neuroepithelial tumors.","method":"In vivo mouse CNS development model (conditional knockdown/KO); co-immunoprecipitation; immunofluorescence localization; ASPP2 heterozygous and homozygous loss-of-function analysis","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP plus in vivo loss-of-function with specific morphological phenotypes; replicated across two independent papers (PMIDs 20619750 and 20619648)","pmids":["20619750"],"is_preprint":false},{"year":2010,"finding":"ASPP2 interacts and colocalizes with PAR-3 at apical cell-cell junctions in polarized epithelial cells; depletion of ASPP2 causes polarity defects (tight junction formation, apical domain development) and mislocalization of PAR-3; disruption of the ASPP2–PAR-3 interaction causes the same polarity defects.","method":"Co-immunoprecipitation; immunofluorescence colocalization; siRNA depletion; domain-interaction mapping","journal":"Current biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP plus RNAi loss-of-function plus interaction-disruption mutants; independently replicated (PMID 20619750)","pmids":["20619648"],"is_preprint":false},{"year":2010,"finding":"PP1A dephosphorylates TAZ at Ser-89 and Ser-311, promotes TAZ nuclear translocation, and stabilizes TAZ by disrupting SCF E3 ubiquitin ligase binding; ASPP2 facilitates the interaction between TAZ and PP1, thereby promoting TAZ dephosphorylation and TAZ-dependent gene expression.","method":"In vitro phosphatase assay; co-immunoprecipitation; phospho-specific antibody Western blot; nuclear/cytoplasmic fractionation; gene reporter assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro phosphatase assay plus Co-IP; single lab with two orthogonal methods","pmids":["21189257"],"is_preprint":false},{"year":2011,"finding":"H. pylori CagA associates with ASPP2 upon translocation into host cells; this CagA–ASPP2 interaction recruits p53 into the complex, inhibits p53 apoptotic function, and leads to enhanced p53 degradation in an ASPP2-dependent manner; CagA-infected cells show increased resistance to doxorubicin-induced apoptosis requiring ASPP2.","method":"Co-immunoprecipitation from infected cells; apoptosis assay with doxorubicin; siRNA depletion of ASPP2; Western blot for p53 levels","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP from infected cells, ASPP2 RNAi epistasis, functional apoptosis readout; multiple orthogonal methods; independently replicated structurally (PMID 24474782)","pmids":["21562218"],"is_preprint":false},{"year":2012,"finding":"ASPP2 N-terminal RAS-association domain binds Ras-GTP at the plasma membrane and stimulates Ras-induced Raf/MEK/ERK signaling by promoting Ras-GTP loading, B-Raf/C-Raf dimerization, and C-Raf phosphorylation; decreased ASPP2 attenuates H-RasV12-induced senescence in normal human fibroblasts and keratinocytes; the short isoform BBP/53BP2S, lacking the N-terminus, fails to bind Ras-GTP or stimulate ERK.","method":"Ras-GTP pull-down; co-immunoprecipitation (Ras-GTP/ASPP2); pERK Western blot; plasma membrane colocalization by confocal microscopy; Raf dimerization assay; senescence assay (β-galactosidase); isoform comparison (53BP2S vs ASPP2)","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — GTP-Ras pull-down, dimerization assay, isoform-specific functional comparison, and in vivo senescence assay; multiple orthogonal methods","pmids":["23248303"],"is_preprint":false},{"year":2013,"finding":"ASPP1 and ASPP2 preferentially bind active (GTP-loaded) RAS via their N-terminal RAS-association domains; ASPP2 co-localizes with RAS at the cellular membrane and contributes to RAS membrane localization; in cancer cells, ASPP1/ASPP2 cooperate with oncogenic RAS to enhance p53 transcriptional apoptotic function.","method":"Pull-down with Ras-GTP (GTP-agarose); co-immunoprecipitation; confocal colocalization; luciferase reporter for p53 transcriptional targets; apoptosis assay","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Ras-GTP pull-down plus Co-IP plus colocalization; single lab corroborating PMID 23248303","pmids":["23392125"],"is_preprint":false},{"year":2013,"finding":"FIH-1 (factor inhibiting HIF-1) hydroxylates ASPP2 at asparagine-986 within the ankyrin repeat domain; this hydroxylation is required for Par-3 binding to ASPP2—FIH-1 depletion impairs Par-3–ASPP2 interaction and causes relocation of ASPP2 from cell-cell contacts to the cytosol—without affecting p53 binding, apoptosis, or proliferation.","method":"Mass spectrometry identification of hydroxylation site; site-directed mutagenesis; co-immunoprecipitation; immunofluorescence localization; siRNA depletion of FIH-1; FIH-1 inhibitor (DMOG)","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1 / Moderate — MS identification of PTM site, mutagenesis confirmation, and Co-IP-based functional consequence; multiple orthogonal methods in one study","pmids":["23606740"],"is_preprint":false},{"year":2013,"finding":"ASPP2 inhibits ΔNp63 expression through its ability to bind IκB and enhance nuclear RelA/p65 (NF-κB), which mediates transcriptional repression of p63; heterozygosity of p63 (but not p53) prevents squamous cell carcinoma development in ASPP2-haploinsufficient mice.","method":"Co-immunoprecipitation (ASPP2–IκB interaction); nuclear fractionation; ChIP for RelA/p65 on p63 promoter; genetic epistasis (p63+/−; p53+/− crosses with ASPP2Δexon3/+ mice); tumor incidence analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP, ChIP, and genetic epistasis in mouse model; multiple orthogonal methods establishing pathway position","pmids":["24127607"],"is_preprint":false},{"year":2013,"finding":"ASPP2 intramolecular autoinhibitory interaction: the disordered proline-rich domain of ASPP2 competes with p53 core domain for binding the n-src loop of the ASPP2 SH3 domain; p53 core domain and NFκB (residues 303–332) bind partially overlapping sites on the ASPP2 SH3 RT loop; Bcl-2 binds ASPP2 at sites largely distinct from p53/NFκB.","method":"Fluorescence anisotropy competition experiments; peptide-based binding studies; recombinant protein assays","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — quantitative biophysical competition experiments with recombinant proteins; single lab extending prior structural work","pmids":["23472201"],"is_preprint":false},{"year":2013,"finding":"ASPP2 is a novel substrate of MAPK (ERK); MAPK phosphorylation of ASPP2 is required for RAS-induced increased binding to p53 and enhanced transactivation of pro-apoptotic genes; a phosphorylation-deficient ASPP2 mutant shows reduced p53 binding and fails to enhance apoptosis.","method":"In vitro MAPK kinase assay; phosphorylation-deficient ASPP2 mutant; co-immunoprecipitation of ASPP2–p53; luciferase reporter assay; apoptosis assay","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay plus mutagenesis plus functional Co-IP; single lab","pmids":["24312625"],"is_preprint":false},{"year":2013,"finding":"ASPP2 attenuates Src kinase activation in a Csk (C-terminal Src kinase)-dependent manner; ASPP2 (but not ASPP1) transfection decreases Src-pY416 phosphorylation; this ASPP2-mediated Src inactivation reduces cell migration.","method":"Transfection of ASPP2 vs ASPP1; Western blot for Src-pY416; Csk siRNA epistasis; wound-healing/migration assay","journal":"Carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Csk RNAi epistasis plus specific phospho-readout; single lab, two methods","pmids":["23671128"],"is_preprint":false},{"year":2013,"finding":"DDA3 (a p53 target oncoprotein) binds ASPP2 via its residues 118–241 to both N- and C-terminal regions of ASPP2; DDA3 dose-dependently inhibits ASPP2-stimulated p53-mediated BAX promoter activation without interfering with ASPP2–p53 binding.","method":"Yeast two-hybrid screen; GST pull-down; immunofluorescence colocalization; domain mapping; luciferase BAX promoter reporter assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — GST pull-down confirmed by reporter functional assay; single lab","pmids":["18793611"],"is_preprint":false},{"year":2014,"finding":"Crystal structure (2.0 Å) of the N-terminal CagA subdomain bound to a 7-kDa proline-rich sequence of ASPP2: CagA forms a three-helix bundle with a deep binding cleft for a 20-aa conserved ASPP2 peptide that adopts an extended helix; structure-based loss-of-contact mutations in either CagA or ASPP2 disrupt the interaction in vitro and in vivo and alter ASPP2 function.","method":"X-ray crystallography (2.0 Å co-crystal); yeast two-hybrid domain delineation; in vitro biochemical binding confirmation; structure-based mutagenesis; functional cell-based assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — atomic resolution co-crystal structure with mutagenesis validation in vitro and in vivo","pmids":["24474782"],"is_preprint":false},{"year":2014,"finding":"ASPP2 induces mesenchymal-to-epithelial transition (MET) via its PAR3-binding N-terminus (independently of p53 binding); mechanistically, ASPP2 prevents β-catenin from transactivating ZEB1 by (i) forming an ASPP2–β-catenin–E-cadherin ternary complex and (ii) inhibiting N-terminal phosphorylation of β-catenin to stabilize the β-catenin–E-cadherin complex; ASPP2 limits oncogenic RAS pro-invasive effects and inhibits tumor metastasis in vivo.","method":"Co-immunoprecipitation (ASPP2–β-catenin–E-cadherin ternary complex); domain-mapping (N-terminus vs. p53-binding domain); β-catenin phosphorylation assay; in vivo mouse kidney MET model; in vivo metastasis assay","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — ternary complex Co-IP, domain-specific rescue, in vivo models; multiple orthogonal methods","pmids":["25344754"],"is_preprint":false},{"year":2014,"finding":"ASPP2 forms an apical-lateral polarity complex at tight junctions acting as a scaffold for PP1 and junctional YAP via dedicated binding domains; ASPP2 directly induces dephosphorylation and activation of junctional YAP; this mechanism controls YAP function in polarized epithelial cells and in the murine colonic epithelium in vivo.","method":"Co-immunoprecipitation (ASPP2–PP1–YAP complex); domain mapping; phospho-YAP Western blot; siRNA depletion; in vivo murine colonic epithelium analysis","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP scaffold complex plus phosphorylation assay plus in vivo; single lab","pmids":["25360797"],"is_preprint":false},{"year":2014,"finding":"ASPP2 induces autophagic apoptosis in hepatoma cells through p53/p73-independent CHOP expression; CHOP decreases Bcl-2 expression, releasing Beclin-1 from Bcl-2–Beclin-1 complexes to initiate autophagy; ASPP2 also induces DRAM expression; CHOP promotes nuclear translocation of Bcl-2 where it is sequestered in ASPP2–Bcl-2 nuclear complexes, preventing Bcl-2 return to the cytoplasm.","method":"Western blot; co-immunoprecipitation (ASPP2–Bcl-2); siRNA knockdown of CHOP; adenoviral ASPP2 overexpression; autophagy and apoptosis assays; nuclear/cytoplasmic fractionation","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus RNAi pathway dissection plus compartment fractionation; single lab","pmids":["25032846"],"is_preprint":false},{"year":2014,"finding":"The E3 ubiquitin ligase Itch mediates ASPP2 degradation and ubiquitination via interaction of the ASPP2 PPXY motif with Itch WW domains; Yap1 competes with Itch for binding to ASPP2 and prevents Itch-mediated ASPP2 degradation, indicating antagonistic regulation of ASPP2 protein stability.","method":"Co-immunoprecipitation; ubiquitination assay in vivo; domain mapping (PPXY–WW interaction); competition binding assay; protein stability (CHX chase)","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ubiquitination assay plus Co-IP competition; single lab","pmids":["25436413"],"is_preprint":false},{"year":2013,"finding":"The ubiquitin E3 ligase Siah2 interacts with ASPP2 and ASPP1 under hypoxic conditions and targets ASPP2 for ubiquitination and proteasomal degradation via degron motifs in ASPP2; Siah2 inhibition increases ASPP2 levels and enhances tight junction integrity and polarity in 3D organotypic culture; hypoxia-induced Siah2 upregulation decreases ASPP2 levels and impairs polarity.","method":"LC-MS/MS identification of Siah2–ASPP2 interaction; co-immunoprecipitation; ubiquitination assay; degron mutant mapping; 3D organotypic culture; siRNA depletion; polarity assays","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS-based interaction identification confirmed by Co-IP and ubiquitination assay with functional polarity readout; single lab","pmids":["23644657"],"is_preprint":false},{"year":2015,"finding":"ASPP2 physically interacts with C-terminal Src kinase (CSK) and stimulates its kinase activity, leading to Src inactivation, AP1-mediated downregulation of Snail expression, and suppression of HCC stemness; pharmacological inhibition of Src attenuates ASPP2-deficiency effects.","method":"Co-immunoprecipitation (ASPP2–CSK); CSK kinase activity assay; Src phosphorylation Western blot; AP1/Snail reporter; tumor sphere formation; side-population assay; Src inhibitor rescue","journal":"Tumour biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus kinase activity assay plus pharmacological rescue; single lab","pmids":["27473084"],"is_preprint":false},{"year":2015,"finding":"An intrinsically disordered region of ASPP2 (residues 448–692) that is unstructured in solution binds the N-terminal domain of CagA; peptide array mapping identified multiple distributed interaction sites throughout ASPP2 for CagA, extending beyond the crystallographically resolved fragment.","method":"SEC-MALS; circular dichroism; NMR; surface plasmon resonance; isothermal titration calorimetry; peptide array mapping","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — multiple biophysical methods (NMR, ITC, SPR, CD) in one rigorous study","pmids":["25963096"],"is_preprint":false},{"year":2015,"finding":"ASPP1 and ASPP2 interact with centrosome linker protein C-Nap1; co-depletion of ASPP1/2 inhibits C-Nap1 re-association with centrosomes at mitotic exit and impairs centrosome linker reassembly; ASPP1/2 facilitate PP1α–C-Nap1 interaction and antagonize NEK2A-mediated C-Nap1 Ser2417/2421 phosphorylation in a PP1-dependent manner.","method":"Co-immunoprecipitation (ASPP2–C-Nap1, ASPP2–PP1α); siRNA co-depletion; phospho-C-Nap1 (Ser2417/2421) Western blot; centrosome immunofluorescence; NEK2A kinase assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus RNAi epistasis plus phospho-readout; single lab","pmids":["25660448"],"is_preprint":false},{"year":2012,"finding":"Crystal structure of the p73 DNA-binding domain (DBD) bound to ASPP2 ankyrin repeat and SH3 domains solved at high resolution; p73 DBD exhibits a divergent L2 loop (two-residue insertion that repacks the p53 R175 hotspot equivalent); ASPP2 binding is preserved via conformational adaptation in both the ankyrin repeat and SH3 domains.","method":"X-ray crystallography (high-resolution crystal structures of free p73 DBD and p73 DBD–ASPP2 Ank-SH3 complex)","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — atomic-resolution crystal structure; single lab but high-quality structural data","pmids":["22917970"],"is_preprint":false},{"year":2017,"finding":"ASPP2 suppresses TGF-β1-induced EMT in gastric cancer cells by interacting with E3 ubiquitin ligase ITCH and inhibiting ITCH-mediated degradation of Smad7 (a negative regulator of TGF-β1–Smad2/3 signaling); ASPP2 promotes PAR3 recruitment to cell-cell junctions.","method":"Co-immunoprecipitation (ASPP2–ITCH); Smad7 degradation assay; TGF-β1–Smad2/3 signaling Western blot; immunofluorescence (PAR3 junctional localization); migration/invasion assay; in vivo peritoneal dissemination model","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus functional degradation assay plus in vivo model; single lab","pmids":["28400336"],"is_preprint":false},{"year":2019,"finding":"ASPP2 interacts with SREBP-2 in the nucleus and restricts SREBP-2 transcriptional activity on mevalonate pathway target genes (including HMGCR); ASPP2 depletion increases cholesterol levels and tumor-initiating capability; simvastatin rescues ASPP2-depletion-induced phenotypes.","method":"Co-immunoprecipitation (ASPP2–SREBP-2 nuclear); ChIP/reporter assay for SREBP-2 targets; cholesterol measurement; gene expression profiling; xenograft model; simvastatin pharmacological rescue","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — nuclear Co-IP plus gene expression profiling plus pharmacological rescue; single lab","pmids":["31685796"],"is_preprint":false},{"year":2016,"finding":"ASPP2 inhibits NF-κB–induced transcription of BECN1 (Beclin-1) directly via an ASPP2–p65/RelA–IκBα complex that inhibits IκBα phosphorylation and p65 nuclear translocation; ASPP2 also binds BECN1 and reshapes the PIK3C3 complex (decreasing PIK3C3–UVRAG interaction, increasing Rubicon binding), thereby inhibiting autophagy initiation.","method":"Co-immunoprecipitation (ASPP2–p65–IκBα complex; ASPP2–BECN1); IκBα phosphorylation assay; BECN1 promoter-reporter assay; PIK3C3 complex pull-down; autophagic flux assay; xenograft model","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple Co-IPs plus promoter assay plus complex reshaping; single lab","pmids":["27929538"],"is_preprint":false},{"year":2020,"finding":"CagA–ASPP2 interaction promotes remodeling of the PAR polarity complex and loss of cell polarity in H. pylori-infected gastric epithelial cells; blockade of ASPP2 signaling by EGFR inhibitors or a CagA-binding ASPP2 peptide prevents polarity loss and decreases H. pylori survival in infected gastric organoids.","method":"Gastric organoid infection model; high-content imaging screen; co-immunoprecipitation/proximity ligation; EGFR inhibitor treatment; synthetic ASPP2 decoy peptide","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — organoid functional model plus pharmacological and peptide rescue; single lab","pmids":["31964836"],"is_preprint":false},{"year":2020,"finding":"Truncated ASPP2 (t-ASPP2, N-terminal truncation) induces actomyosin relaxation via interaction with PP1 to enable survival of E-cadherin-deficient mammary epithelial cells on stiff matrices (required for ILC initiation); separately, t-ASPP2-induced YAP activation (not actomyosin relaxation) contributes to tumor growth and progression.","method":"Mouse ILC model with E-cadherin loss; actomyosin tension assay; PP1 interaction domain mapping; YAP activity assay; conditional mouse genetics (PTEN loss)","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetically engineered mouse model with mechanistic domain mapping and pathway dissection; single lab","pmids":["32060147"],"is_preprint":false},{"year":2019,"finding":"An alternatively spliced ASPP2 isoform, ASPP2κ (exon skipping generating truncated C-terminus lacking p53-binding sites), acts as a dominant-negative variant impairing TP53-dependent apoptosis induction; ASPP2κ expression causes perturbed proliferation, impaired apoptosis, mitotic failure, and chromosomal instability; its expression is stress-inducible.","method":"mRNA splice variant screening; isoform-specific PCR and epitope-specific antibody generation; forced expression and isoform-specific RNAi in cell models; apoptosis, proliferation, and chromosomal instability assays","journal":"EBioMedicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — isoform-specific KD/OE with defined molecular and cellular phenotypes; single lab","pmids":["30952616"],"is_preprint":false},{"year":2021,"finding":"ASPP2 binds HSF1 in the cytoplasm of HBV-infected cells, preventing HSF1 nuclear translocation and thereby inhibiting transcriptional activation of ATG7; by suppressing ATG7 expression, ASPP2 reduces HBV-induced hepatocyte autophagy and inhibits HBV replication.","method":"Co-immunoprecipitation (ASPP2–HSF1); nuclear/cytoplasmic fractionation; ChIP for HSF1 on ATG7 promoter; Western blot for ATG7; autophagic flux assay; HBV replication assay","journal":"Journal of cellular and molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus nuclear fractionation plus ChIP; single lab with multiple methods","pmids":["34085409"],"is_preprint":false},{"year":2021,"finding":"ATF4 transcription factor binds the TP53BP2 promoter (ChIP-validated) and drives TP53BP2 expression during ischemia/reperfusion stress; TP53BP2 overexpression promotes inflammation and apoptosis in hepatocytes, and sufentanil protection is mediated by suppression of ATF4-driven TP53BP2 induction.","method":"ChIP assay (ATF4 on TP53BP2 promoter); TP53BP2 overexpression in H/R cell model; Western blot; ELISA for inflammatory markers; in vivo rat I/R model","journal":"Inflammation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP identification of transcriptional regulator plus functional overexpression assay; single lab","pmids":["33751357"],"is_preprint":false},{"year":2009,"finding":"DEAD-box protein Ddx42p physically interacts with ASPP2 via the Ddx42p C-terminal region and ASPP2 mid-N-terminal + Ank-SH3 domains; Ddx42p overexpression interferes with ASPP2-induced apoptosis; elevated Ddx42p shifts ASPP2 localization from nucleus+cytoplasm to predominantly cytoplasm.","method":"Co-immunoprecipitation; domain mapping; apoptosis assay with Ddx42p overexpression/knockdown; immunofluorescence localization","journal":"Oncogene","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP with localization; single lab, limited validation","pmids":["19377511"],"is_preprint":false},{"year":2023,"finding":"TP53BP2 downregulates SOCS2 expression, thereby facilitating JAK/STAT signaling and enhancing the anti-HBV effect of IFN-α in hepatocytes.","method":"In vitro and in vivo experiments; SOCS2 expression measurement upon TP53BP2 loss/gain; JAK/STAT pathway readouts; antiviral assay with IFN-α","journal":"Journal of hepatology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — functional pathway assay in single study without detailed mechanistic dissection of direct interaction","pmids":["37858684"],"is_preprint":false}],"current_model":"ASPP2 (TP53BP2) is a multifunctional scaffold protein whose C-terminal ankyrin repeats and SH3 domain directly bind the p53 (and p63/p73) DNA-binding domain, selectively stimulating transcription of pro-apoptotic target genes (Bax, PUMA, PIG3) over cell-cycle arrest genes; this interaction is regulated by an intramolecular autoinhibitory mechanism involving the natively disordered N-terminal proline-rich domain, by MAPK phosphorylation (which enhances p53 binding), by proteasomal degradation (controlled by the E3 ligases Itch and Siah2), and by FIH-1-mediated hydroxylation of Asn-986; the same Ank-SH3 domain also binds Bcl-2 (via the BH4 domain), NF-κB p65/RelA (in an IκB-like manner), and APP-BP1; independently of p53, ASPP2 regulates epithelial polarity by binding PAR-3 (interaction dependent on FIH-1 hydroxylation) and acting as a PP1 scaffold that dephosphorylates and activates junctional YAP/TAZ; its N-terminal RAS-association domain binds active Ras-GTP, potentiates Raf/MEK/ERK signaling to promote oncogene-induced senescence, and is required for cooperation with oncogenic RAS to enhance p53-dependent apoptosis; ASPP2 also suppresses the mevalonate pathway by binding nuclear SREBP-2, inhibits autophagy by reshaping the PIK3C3–Beclin-1 complex and by sequestering HSF1 to prevent ATG7 transcription, and represses p63 expression via NF-κB–mediated transcriptional suppression; the H. pylori oncoprotein CagA exploits the ASPP2 proline-rich domain to form a high-affinity complex that subverts ASPP2 polarity and apoptosis functions."},"narrative":{"mechanistic_narrative":"ASPP2 (TP53BP2) is a multidomain scaffold protein that couples the p53 family to programmed cell death and links cell polarity, RAS signaling, and metabolic control to tumor suppression [PMID:8875926, PMID:14729977, PMID:23248303]. Its C-terminal ankyrin repeats and SH3 domain dock onto the DNA-binding domains of p53, p63, and p73 — engaging the L2 and L3 loops of the p53 core in a mode that overlaps the DNA-binding surface, such that the six most frequent cancer-associated p53 mutations disrupt the interaction and ASPP2 binding is mutually exclusive with response-element DNA binding [PMID:8875926, PMID:16887812, PMID:18676979, PMID:22917970]. Through this interface ASPP2 selectively stimulates transcription of pro-apoptotic targets (Bax, PUMA, PIG3) rather than cell-cycle arrest genes, and much of its p53-independent apoptotic output is relayed through p63/p73 [PMID:14729977]. The same Ank-SH3 module binds Bcl-2 family proteins at the conserved BH4 motif and engages NF-κB p65/RelA, while a natively disordered N-terminal proline-rich domain folds back to autoinhibit Ank-SH3, gating partner access [PMID:8668206, PMID:18719108, PMID:18448430, PMID:23472201]. ASPP2 activity is tuned by MAPK/ERK phosphorylation that enhances p53 binding upon RAS activation, by FIH-1 hydroxylation of Asn-986 that licenses Par-3 binding, and by proteasomal turnover through the E3 ligases Itch and Siah2 [PMID:24312625, PMID:23606740, PMID:25436413, PMID:23644657]. Beyond apoptosis, ASPP2 acts at apical cell-cell junctions: it binds Par-3 to maintain epithelial and neuroepithelial polarity and scaffolds PP1 to dephosphorylate and activate junctional YAP/TAZ [PMID:20619750, PMID:20619648, PMID:21189257, PMID:25360797]. Its N-terminal RAS-association domain binds Ras-GTP at the membrane, potentiating Raf/MEK/ERK signaling and oncogene-induced senescence and cooperating with oncogenic RAS to enhance p53-dependent apoptosis [PMID:23248303, PMID:23392125]. ASPP2 further suppresses tumorigenic programs by restraining ΔNp63 via NF-κB, blocking β-catenin-driven ZEB1 to promote mesenchymal-to-epithelial transition, inhibiting autophagy initiation, and restricting nuclear SREBP-2 control of the mevalonate pathway [PMID:24127607, PMID:25344754, PMID:27929538, PMID:31685796]. The H. pylori oncoprotein CagA exploits the disordered ASPP2 proline-rich region to form a high-affinity complex that subverts ASPP2 polarity and apoptotic functions [PMID:21562218, PMID:24474782, PMID:31964836].","teleology":[{"year":1996,"claim":"Established the structural basis for how ASPP2 recognizes p53 and why the interaction is tumor-relevant, answering how a non-DNA-binding partner engages the p53 core.","evidence":"X-ray crystallography of the p53 core–53BP2 complex with cancer-mutant in vitro binding assays","pmids":["8875926"],"confidence":"High","gaps":["Did not show whether binding occurs on or off DNA in cells","No functional transcriptional consequence quantified"]},{"year":1996,"claim":"Showed ASPP2 also binds Bcl-2 through the same Ank-SH3 domains and that Bcl-2 and p53 compete, raising the possibility of partner-switching control of apoptosis.","evidence":"Yeast two-hybrid, in vitro GST pull-down with competition assays, immunofluorescence, flow cytometry","pmids":["8668206"],"confidence":"High","gaps":["Physiological relevance of competition not established in vivo","Cytoplasmic colocalization only partial"]},{"year":1998,"claim":"Resolved the puzzle that cytoplasmic ASPP2 still enhances p53 transcriptional output, framing it as a signal-transduction modulator rather than an obligatory nuclear cofactor.","evidence":"Immunofluorescence localization plus p53-dependent reporter and target-protein assays","pmids":["9748285"],"confidence":"Medium","gaps":["Mechanism connecting cytoplasmic ASPP2 to nuclear p53 unresolved","Single-lab observation"]},{"year":1999,"claim":"Identified NF-κB p65/RelA as a direct ASPP2 partner that suppresses ASPP2-induced apoptosis and mapped apoptotic activity to the full-length protein.","evidence":"Yeast two-hybrid, in vitro and mammalian two-hybrid, GFP localization, transfection apoptosis assay","pmids":["10498867"],"confidence":"Medium","gaps":["Domain of p65 binding not precisely mapped here","p53-dependence of the apoptotic effect not isolated"]},{"year":2000,"claim":"Placed ASPP2 in the DNA-damage response by showing UV induces its protein in a p53-independent manner while wild-type p53 suppresses it, defining a feedback relationship that lowers the apoptotic threshold.","evidence":"Western blot in defined-genotype lines, inducible expression, antisense knockdown, clonogenic and apoptosis assays","pmids":["11027272"],"confidence":"High","gaps":["Mechanism of p53-mediated ASPP2 suppression not defined","Post-transcriptional vs transcriptional control unclear"]},{"year":2002,"claim":"Defined the central functional principle: ASPPs selectively drive apoptotic over arrest p53 targets and extend this to p63/p73, with p63/p73 carrying p53-independent apoptosis.","evidence":"Co-IP, in vitro binding, promoter-selective luciferase reporters, p63/p73 RNAi epistasis, apoptosis assays","pmids":["14729977"],"confidence":"High","gaps":["Molecular basis of promoter selectivity not structurally explained","How ASPP2 distinguishes apoptotic from arrest promoters unknown"]},{"year":2003,"claim":"Connected ASPP2 to neddylation control by showing it binds APP-BP1 and inhibits NEDD8 conjugation to Cullin-1, with consequences for proliferation and neuronal apoptosis.","evidence":"Endogenous Co-IP, domain mapping, NEDD8 conjugation assay, proliferation and apoptosis assays","pmids":["12694406"],"confidence":"Medium","gaps":["Integration with p53/apoptotic functions unclear","Single-lab finding"]},{"year":2004,"claim":"Defined gene architecture (53BP2S short and 53BP2L/ASPP2 long isoforms) by alternative splicing, providing the basis for isoform-specific functions.","evidence":"RT-PCR, genomic cloning, sequencing of TP53BP2 transcripts","pmids":["14766226"],"confidence":"Medium","gaps":["Functional differences between isoforms not assessed here"]},{"year":2004,"claim":"Showed HCV core protein competes with p53 for ASPP2, an early example of viral subversion of ASPP2-enhanced apoptosis.","evidence":"Yeast two-hybrid, in vitro competition binding, apoptosis and reporter assays","pmids":["14985081"],"confidence":"Medium","gaps":["Binding site on ASPP2 not mapped","In-cell relevance during infection not tested"]},{"year":2005,"claim":"Multiple studies defined ASPP2 regulatory inputs and outputs: E2F transcriptional induction in S-phase, proteasomal stability control, the mutually exclusive p53/DNA-binding mechanism, Mdm2/MdmX antagonism, and a mitochondrial apoptotic pathway.","evidence":"ChIP and promoter reporters; proteasome inhibition and ubiquitination assays; biophysical binding (SPR/ITC/anisotropy) with p53 mutant panels; mutant dissection; subcellular fractionation with caspase-9 assays","pmids":["15592436","15731768","16091363","16887812","15782125","15743414"],"confidence":"High","gaps":["E3 ligase mediating ASPP2 ubiquitination not yet identified in this period","How cytoplasmic/mitochondrial ASPP2 ties to nuclear p53 transcription unresolved"]},{"year":2008,"claim":"Quantitative biophysics and NMR established the autoinhibitory architecture and binding thermodynamics: the disordered Pro domain competes with Ank-SH3 for partner peptides, and ASPP2 binds Bcl-2 family BH4 motifs and p53/p63/p73 DBDs at low-micromolar 1:1 stoichiometry, with ASPP2 uniquely binding PUMA DNA.","evidence":"CD, NMR, SEC, fluorescence anisotropy, peptide arrays, SPR, ITC, EMSA, homology modeling","pmids":["18719108","18676979","18448430"],"confidence":"High","gaps":["Trigger that relieves intramolecular autoinhibition in cells not defined","Functional impact of PUMA-DNA binding on transcription unresolved"]},{"year":2008,"claim":"Identified DDA3 as a p53-target oncoprotein that binds ASPP2 and inhibits its stimulation of p53-mediated BAX activation without blocking ASPP2–p53 binding.","evidence":"Yeast two-hybrid, GST pull-down, colocalization, domain mapping, BAX reporter assay","pmids":["18793611"],"confidence":"Medium","gaps":["Mechanism of inhibition without disrupting p53 binding unclear","Single-lab finding"]},{"year":2009,"claim":"Revealed a p53-independent role in epithelial/neuroepithelial polarity by showing ASPP2 binds Par-3 and maintains junctions, with loss causing neuroepithelial tumor-like phenotypes in vivo.","evidence":"In vivo mouse CNS loss-of-function, Co-IP, immunofluorescence","pmids":["20619750"],"confidence":"High","gaps":["Biochemical determinants of the Par-3 interaction not mapped here","Link between polarity loss and tumorigenesis mechanistic at this stage"]},{"year":2010,"claim":"Extended the polarity role to epithelial cells and mechanistically linked ASPP2 to YAP/TAZ regulation, showing it scaffolds PP1 to dephosphorylate TAZ and facilitates the Par-3 apical complex.","evidence":"Co-IP, immunofluorescence, siRNA depletion, in vitro phosphatase assay, fractionation, reporter assays","pmids":["20619648","21189257"],"confidence":"Medium","gaps":["How polarity and apoptotic functions are coordinated unclear","PP1 scaffolding stoichiometry not defined"]},{"year":2012,"claim":"Defined the N-terminal RAS-association function: ASPP2 binds Ras-GTP at the membrane, potentiates Raf/MEK/ERK signaling and oncogene-induced senescence, distinguishing the long isoform from senescence-incompetent 53BP2S.","evidence":"Ras-GTP pull-down, Co-IP, pERK Western, confocal colocalization, Raf dimerization, senescence assay, isoform comparison","pmids":["23248303"],"confidence":"High","gaps":["How RAS engagement feeds back to p53/apoptosis not yet linked here","Structural basis of RA-domain–Ras binding not solved"]},{"year":2012,"claim":"Crystallography of the p73 DBD–ASPP2 Ank-SH3 complex showed conformational adaptation accommodates the divergent p73 L2 loop, explaining conserved binding across the p53 family.","evidence":"X-ray crystallography of free and ASPP2-bound p73 DBD","pmids":["22917970"],"confidence":"High","gaps":["Affinity differences across family members not functionally resolved here"]},{"year":2013,"claim":"A cluster of studies defined ASPP2 post-translational and signaling regulation: FIH-1 hydroxylation of Asn-986 gating Par-3 binding, MAPK phosphorylation enhancing p53 binding upon RAS activation, NF-κB-mediated ΔNp63 repression as a tumor-suppressive axis, mapped competing autoinhibitory/partner sites on the SH3, Csk-dependent Src inactivation, Siah2-mediated hypoxic degradation, and RAS cooperation to enhance p53 apoptosis.","evidence":"MS PTM mapping with mutagenesis and Co-IP; in vitro kinase assays with phospho-mutants; Co-IP/ChIP and mouse genetic epistasis; biophysical competition; Csk RNAi epistasis with migration; LC-MS/MS, ubiquitination and 3D polarity assays; Ras-GTP pull-down with apoptosis reporters","pmids":["23606740","24312625","24127607","23472201","23671128","23644657","23392125"],"confidence":"High","gaps":["Crosstalk between hydroxylation, phosphorylation, and degradation inputs not integrated","Stimulus-specific deployment of distinct ASPP2 functions unresolved"]},{"year":2014,"claim":"Defined the CagA–ASPP2 hijacking mechanism structurally and functionally, and expanded ASPP2 tumor-suppressive functions to MET, YAP/PP1 junctional scaffolding, autophagic apoptosis via CHOP-Bcl-2-Beclin-1, and Itch-mediated degradation antagonized by Yap1.","evidence":"2.0 Å co-crystal with mutagenesis; Co-IP of ternary β-catenin/E-cadherin complex with in vivo MET/metastasis models; PP1–YAP scaffold Co-IP with in vivo colonic epithelium; CHOP RNAi pathway dissection; PPXY–WW ubiquitination and competition assays","pmids":["24474782","25344754","25360797","25032846","25436413"],"confidence":"High","gaps":["Hierarchy among ASPP2 polarity, MET, and apoptotic outputs unclear","Whether these functions operate in the same cell context not established"]},{"year":2015,"claim":"Refined CagA binding to a distributed disordered ASPP2 region, defined ASPP2–Csk–Src–Snail suppression of HCC stemness, and identified an ASPP1/2–PP1α–C-Nap1 axis in centrosome linker reassembly.","evidence":"SEC-MALS/NMR/SPR/ITC/peptide arrays; Co-IP with Csk kinase and AP1/Snail reporters and stemness assays; Co-IP with phospho-C-Nap1 and centrosome imaging","pmids":["25963096","27473084","25660448"],"confidence":"Medium","gaps":["Mitotic centrosome role not connected to apoptotic/polarity functions","Csk-stimulating mechanism not structurally defined"]},{"year":2017,"claim":"Showed ASPP2 suppresses TGF-β1-induced EMT by stabilizing Smad7 through inhibition of ITCH-mediated degradation, integrating ASPP2 into junctional and TGF-β anti-tumor control.","evidence":"Co-IP, Smad7 degradation assay, Smad2/3 Western, immunofluorescence, migration/invasion and in vivo dissemination","pmids":["28400336"],"confidence":"Medium","gaps":["Relationship between ASPP2–ITCH stabilization of Smad7 and Itch-mediated ASPP2 degradation unclear","Single-lab finding"]},{"year":2019,"claim":"Linked ASPP2 to lipid metabolism by showing it restrains nuclear SREBP-2 control of the mevalonate pathway, and identified a dominant-negative splice isoform (ASPP2κ) that impairs p53 apoptosis and drives chromosomal instability.","evidence":"Nuclear Co-IP, ChIP/reporter, cholesterol measurement, xenograft and simvastatin rescue; isoform-specific KD/OE with CIN assays","pmids":["31685796","30952616"],"confidence":"Medium","gaps":["Direct vs indirect SREBP-2 inhibition mechanism not fully resolved","Regulation of ASPP2κ splicing under stress not detailed"]},{"year":2020,"claim":"Defined CagA-driven PAR-complex remodeling in gastric organoids and a truncated ASPP2–PP1 actomyosin/YAP mechanism enabling E-cadherin-deficient epithelial survival in lobular carcinoma.","evidence":"Organoid infection with high-content imaging, EGFR-inhibitor and decoy-peptide rescue; mouse ILC model with PP1 domain mapping and YAP dissection","pmids":["31964836","32060147"],"confidence":"Medium","gaps":["Generality of t-ASPP2 mechanism beyond ILC unclear","How truncation alters ASPP2 partner selection not detailed"]},{"year":2021,"claim":"Established autophagy/transcription control roles in viral and stress contexts: ASPP2 sequesters HSF1 to block ATG7 transcription and limit HBV-induced autophagy, and is itself an ATF4 transcriptional target driving inflammation and apoptosis during ischemia/reperfusion.","evidence":"Co-IP, fractionation and ChIP for HSF1–ATG7; ChIP of ATF4 on TP53BP2 promoter with overexpression and in vivo I/R models","pmids":["34085409","33751357"],"confidence":"Medium","gaps":["Direct ASPP2 contact residues on HSF1 not mapped","Integration with NF-κB/Beclin-1 autophagy control unclear"]},{"year":2016,"claim":"Defined dual autophagy suppression: ASPP2 inhibits NF-κB-driven BECN1 transcription via an ASPP2–p65–IκBα complex and reshapes the PIK3C3–Beclin-1 complex to block autophagy initiation.","evidence":"Co-IP, IκBα phosphorylation assay, BECN1 promoter reporter, PIK3C3 complex pull-down, autophagic flux, xenograft","pmids":["27929538"],"confidence":"Medium","gaps":["Balance between pro-autophagic (CHOP axis) and anti-autophagic ASPP2 effects unresolved","Single-lab finding"]},{"year":null,"claim":"How ASPP2's distinct modular functions — p53-family apoptosis, RAS signaling, junctional polarity/YAP, autophagy and metabolic control — are coordinated and selectively deployed within a single cell remains unresolved.","evidence":"No single study integrates the autoinhibition, PTM, and partner-switching inputs into a unified regulatory logic","pmids":[],"confidence":"Medium","gaps":["No structure of full-length ASPP2 with multiple partners","Stimulus-to-function mapping incomplete","Role of low-confidence partners (Ddx42p, SOCS2 axis) not validated"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[17,18,19,31,37,21]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[5,11,14,16,6]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[5,24,40]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[15]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[2,3,23,45]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[40,47,32]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[21,22,17,18]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[13]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[37]}],"pathway":[{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[0,5,13,4]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[21,22,26,19]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[5,24,40]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[41,32,45]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[10,33,34,6]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[17,18,30]}],"complexes":["ASPP2–PP1–YAP junctional polarity complex","ASPP2–p65/RelA–IκBα complex","ASPP2–β-catenin–E-cadherin complex","PIK3C3–Beclin-1 complex"],"partners":["TP53","TP73","BCL2","RELA","PARD3","RAS","PPP1CA","ITCH"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q13625","full_name":"Apoptosis-stimulating of p53 protein 2","aliases":["Bcl2-binding protein","Bbp","Renal carcinoma antigen NY-REN-51","Tumor suppressor p53-binding protein 2","53BP2","p53-binding protein 2","p53BP2"],"length_aa":1128,"mass_kda":125.6,"function":"Regulator that plays a central role in regulation of apoptosis and cell growth via its interactions with proteins such as TP53 (PubMed:12524540). Regulates TP53 by enhancing the DNA binding and transactivation function of TP53 on the promoters of proapoptotic genes in vivo. Inhibits the ability of NAE1 to conjugate NEDD8 to CUL1, and thereby decreases NAE1 ability to induce apoptosis. Impedes cell cycle progression at G2/M. Its apoptosis-stimulating activity is inhibited by its interaction with DDX42","subcellular_location":"Cytoplasm, perinuclear region; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q13625/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TP53BP2","classification":"Not Classified","n_dependent_lines":76,"n_total_lines":1208,"dependency_fraction":0.06291390728476821},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/TP53BP2","total_profiled":1310},"omim":[{"mim_id":"613369","title":"DEAD-BOX HELICASE 42; DDX42","url":"https://www.omim.org/entry/613369"},{"mim_id":"606455","title":"PROTEIN PHOSPHATASE 1, REGULATORY SUBUNIT 13B; PPP1R13B","url":"https://www.omim.org/entry/606455"},{"mim_id":"605230","title":"TUMOR PROTEIN p53-BINDING PROTEIN 1; TP53BP1","url":"https://www.omim.org/entry/605230"},{"mim_id":"602143","title":"TUMOR PROTEIN p53-BINDING PROTEIN 2; TP53BP2","url":"https://www.omim.org/entry/602143"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Cell Junctions","reliability":"Enhanced"},{"location":"Cytosol","reliability":"Enhanced"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TP53BP2"},"hgnc":{"alias_symbol":["PPP1R13A","ASPP2","53BP2"],"prev_symbol":[]},"alphafold":{"accession":"Q13625","domains":[{"cath_id":"3.10.20.90","chopping":"5-82","consensus_level":"high","plddt":83.8279,"start":5,"end":82},{"cath_id":"2.30.30.40","chopping":"1057-1128","consensus_level":"medium","plddt":89.3306,"start":1057,"end":1128}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13625","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q13625-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q13625-F1-predicted_aligned_error_v6.png","plddt_mean":58.97},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TP53BP2","jax_strain_url":"https://www.jax.org/strain/search?query=TP53BP2"},"sequence":{"accession":"Q13625","fasta_url":"https://rest.uniprot.org/uniprotkb/Q13625.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q13625/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13625"}},"corpus_meta":[{"pmid":"8875926","id":"PMC_8875926","title":"Structure 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ATG7.","date":"2021","source":"Journal of cellular and molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/34085409","citation_count":9,"is_preprint":false},{"pmid":"27625111","id":"PMC_27625111","title":"ASPP2 involvement in p53-mediated HIV-1 envelope glycoprotein gp120 neurotoxicity in mice cerebrocortical neurons.","date":"2016","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/27625111","citation_count":8,"is_preprint":false},{"pmid":"15782125","id":"PMC_15782125","title":"Mdm2 and mdmX prevent ASPP1 and ASPP2 from stimulating p53 without targeting p53 for degradation.","date":"2005","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/15782125","citation_count":8,"is_preprint":false},{"pmid":"25660448","id":"PMC_25660448","title":"The tumor suppressor proteins ASPP1 and ASPP2 interact with C-Nap1 and regulate centrosome linker reassembly.","date":"2015","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/25660448","citation_count":8,"is_preprint":false},{"pmid":"31186708","id":"PMC_31186708","title":"TP53BP2 decreases cell proliferation and induces autophagy in neuroblastoma cell lines.","date":"2019","source":"Oncology letters","url":"https://pubmed.ncbi.nlm.nih.gov/31186708","citation_count":7,"is_preprint":false},{"pmid":"36340606","id":"PMC_36340606","title":"ASPP2 promotes cell apoptosis in cervical cancer through inhibiting autophagy.","date":"2022","source":"Experimental and therapeutic medicine","url":"https://pubmed.ncbi.nlm.nih.gov/36340606","citation_count":6,"is_preprint":false},{"pmid":"24179515","id":"PMC_24179515","title":"Nucleostemin and ASPP2 expression is correlated with pituitary adenoma proliferation.","date":"2013","source":"Oncology letters","url":"https://pubmed.ncbi.nlm.nih.gov/24179515","citation_count":6,"is_preprint":false},{"pmid":"25963096","id":"PMC_25963096","title":"An Intrinsically Disordered Region in the Proapoptotic ASPP2 Protein Binds to the Helicobacter pylori Oncoprotein CagA.","date":"2015","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/25963096","citation_count":6,"is_preprint":false},{"pmid":"25436413","id":"PMC_25436413","title":"The E3 ubiquitin ligase Itch and Yap1 have antagonistic roles in the regulation of ASPP2 protein stability.","date":"2014","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/25436413","citation_count":6,"is_preprint":false},{"pmid":"27447114","id":"PMC_27447114","title":"ASPP2 deficiency causes features of 1q41q42 microdeletion syndrome.","date":"2016","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/27447114","citation_count":6,"is_preprint":false},{"pmid":"28150229","id":"PMC_28150229","title":"Identification of TP53BP2 as a Novel Candidate Gene for Primary Open Angle Glaucoma by Whole Exome Sequencing in a Large Multiplex Family.","date":"2017","source":"Molecular neurobiology","url":"https://pubmed.ncbi.nlm.nih.gov/28150229","citation_count":6,"is_preprint":false},{"pmid":"21183427","id":"PMC_21183427","title":"Cell type specific expression of the apoptosis stimulating protein (ASPP-2) in human tissues.","date":"2010","source":"Acta microbiologica et immunologica Hungarica","url":"https://pubmed.ncbi.nlm.nih.gov/21183427","citation_count":6,"is_preprint":false},{"pmid":"33558702","id":"PMC_33558702","title":"Knockout of ASPP2 promotes DEN-induced hepatocarcinogenesis via the NF-κB pathway in mice.","date":"2021","source":"Cancer gene therapy","url":"https://pubmed.ncbi.nlm.nih.gov/33558702","citation_count":5,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":50762,"output_tokens":13225,"usd":0.175331,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":24874,"output_tokens":8310,"usd":0.16606,"stage2_stop_reason":"end_turn"},"total_usd":0.341391,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1996,\n      \"finding\": \"Crystal structure of the p53 core domain bound to 53BP2 (ASPP2 C-terminal fragment) revealed that the SH3 domain of 53BP2 binds the L3 loop of p53 in a manner distinct from canonical SH3-polyproline complexes, and an ankyrin repeat binds the L2 loop of p53; the binding site overlaps the p53 DNA-binding surface and the six most frequent cancer-associated p53 mutations disrupt 53BP2 binding in vitro.\",\n      \"method\": \"X-ray crystallography (crystal structure of p53 core domain–53BP2 complex); in vitro binding assays with cancer-associated p53 mutants\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — atomic-resolution crystal structure with functional in vitro validation; foundational structural paper replicated by subsequent studies\",\n      \"pmids\": [\"8875926\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"53BP2/ASPP2 interacts with Bcl-2 via its ankyrin repeats and SH3 domain (same domains that mediate p53 binding); Bcl-2 and p53 compete for binding to 53BP2 in vitro; overexpressed 53BP2 partially colocalizes with Bcl-2 in the cytoplasm and increases the proportion of cells at G2/M.\",\n      \"method\": \"Yeast two-hybrid screen; in vitro GST pull-down with bacterially expressed proteins; competition binding assays; immunofluorescence colocalization; cell cycle analysis by flow cytometry\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal yeast two-hybrid plus in vitro reconstitution with competition experiments, replicated in subsequent work\",\n      \"pmids\": [\"8668206\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"53BP2 localizes exclusively to the cytoplasm (not altered by co-expression of wild-type p53); despite this, both 53BP1 and 53BP2 enhance p53-mediated transcriptional activation in cell-based reporter assays, suggesting they function in signal transduction pathways to promote p53 activity without forming a concurrent DNA-binding complex with p53.\",\n      \"method\": \"Immunofluorescence subcellular localization; p53-dependent transcriptional reporter assays; Western blot for p53 target protein induction\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct localization experiment and reporter assay from single lab with two orthogonal methods\",\n      \"pmids\": [\"9748285\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"NF-κB p65 (RelA) subunit directly binds 53BP2/ASPP2 via yeast two-hybrid and in vitro pull-down; co-expression of p65 significantly inhibits 53BP2-induced apoptosis; full-length GFP-53BP2 localizes to perinuclear cytoplasmic puncta and induces apoptosis, whereas N-terminal or C-terminal fragments alone do not.\",\n      \"method\": \"Yeast two-hybrid; in vitro pull-down assay; mammalian two-hybrid assay; GFP fusion subcellular localization; apoptosis assay by transfection\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid confirmed by independent in vitro pull-down and mammalian two-hybrid; single lab\",\n      \"pmids\": [\"10498867\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Endogenous 53BP2/ASPP2 protein levels increase following UV-irradiation-induced DNA damage in a p53-independent manner; conversely, wild-type (but not mutant) p53 suppresses 53BP2 steady-state protein levels; conditional expression of 53BP2 lowers the apoptotic threshold after UV irradiation, and antisense attenuation of 53BP2 induction enhances clonogenic survival.\",\n      \"method\": \"Western blot of endogenous protein in cell lines with defined p53 genotypes; tetracycline-regulated p53 expression system; ecdysone-regulated 53BP2 stable cell lines; antisense oligonucleotide knockdown; clonogenic survival assay; apoptosis assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal genetic approaches (KO cell lines, inducible OE, antisense KD) with defined phenotypic readouts; single lab but rigorous\",\n      \"pmids\": [\"11027272\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"ASPP1 and ASPP2 selectively stimulate the apoptotic transcriptional function of p53 (activating Bax, PIG3, PUMA promoters) but not cell-cycle arrest targets (mdm2, p21); they also bind p63 and p73 in vitro and in vivo and stimulate their apoptotic function; RNAi depletion of endogenous p63/p73 demonstrated that the p53-independent apoptotic activity of ASPP1/ASPP2 is mainly mediated by p63/p73.\",\n      \"method\": \"Co-immunoprecipitation (in vivo); in vitro binding; luciferase reporter assays on apoptotic vs. cell-cycle promoters; RNA interference knockdown of p63/p73; apoptosis assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP plus RNAi epistasis plus promoter-selective reporter assays; multiple orthogonal methods in one rigorous study\",\n      \"pmids\": [\"14729977\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"ASPP2 specifically interacts with APP-BP1 (the NEDD8-activating enzyme subunit) in non-transfected cells through the N-terminal domain ASPP2(332–483); ASPP2 inhibits APP-BP1-mediated NEDD8 conjugation to Cullin-1, reduces APP-BP1-induced cell proliferation, and blocks APP-BP1-triggered apoptosis in primary neurons; ASPP2 also activates NF-κB transcriptional activity.\",\n      \"method\": \"Co-immunoprecipitation from non-transfected cells; domain mapping; NEDD8 conjugation assay (in vitro/cellular); cell proliferation and neuronal apoptosis assays\",\n      \"journal\": \"Journal of neurochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — endogenous Co-IP plus functional neddylation assay; single lab, two orthogonal methods\",\n      \"pmids\": [\"12694406\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Hepatitis C virus core protein interacts with 53BP2/ASPP2 in a yeast two-hybrid assay; the core protein competes with p53 for binding to ASPP2 in vitro and, when co-expressed, inhibits ASPP2-enhanced p53-mediated apoptosis without affecting p53 transcriptional activity on Bax or p21 promoters.\",\n      \"method\": \"Yeast two-hybrid; in vitro competition binding assay; apoptosis assay; reporter assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid confirmed by in vitro competition and cellular apoptosis assay; single lab\",\n      \"pmids\": [\"14985081\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"The TP53BP2 gene encodes two protein isoforms—53BP2S (short, 1005 aa) and 53BP2L/ASPP2 (long, 1128 aa, with an additional N-terminal 123 aa encoded by exon 3)—generated by alternative splicing.\",\n      \"method\": \"RT-PCR and genomic cloning of TP53BP2 transcripts; sequencing\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Strong — direct transcript sequencing from multiple cell lines and tissues; independently confirmed in subsequent studies\",\n      \"pmids\": [\"14766226\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"ASPP2/53BP2L is a transcriptional target of E2F: E2F-1, -2, and -3 bind the endogenous ASPP2 promoter (demonstrated by ChIP), and ectopic E2F-1 increases endogenous ASPP2 mRNA and protein; ASPP2 expression is maximal in early S-phase.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP); luciferase promoter-reporter assays with E2F binding site mutants; Western blot and RT-PCR of endogenous protein after E2F-1 induction; cell-cycle-staged expression analysis\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — ChIP of endogenous promoter plus mutational promoter analysis plus endogenous protein induction; two independent papers (PMIDs 15592436 and 15731768) replicate the finding\",\n      \"pmids\": [\"15592436\", \"15731768\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"ASPP2/53BP2L protein is subject to proteasomal degradation; proteasomal inhibitors (including bortezomib) and anthracycline-based chemotherapy increase ASPP2 protein but not mRNA levels by stabilizing the protein; the central region of ASPP2 is ubiquitinated; siRNA knockdown of ASPP2 attenuates bortezomib-induced apoptosis, particularly in p53 wild-type cells.\",\n      \"method\": \"Proteasome inhibitor treatment; cycloheximide chase (protein half-life); ubiquitination assay; siRNA knockdown; apoptosis assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — protein stability assay combined with ubiquitination detection and functional knockdown; single lab\",\n      \"pmids\": [\"16091363\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"53BP2 binding to p53 and DNA binding are mutually exclusive (no ternary complex detected for GADD45, p21, Bax, or PIG3 response elements); multiple oncogenic p53 mutations (R181E, G245S, R249S, R273H) differentially affect DNA and 53BP2 binding.\",\n      \"method\": \"Biophysical binding assays (fluorescence anisotropy, isothermal titration calorimetry, SPR); competition experiments with DNA response elements and recombinant proteins; p53 mutant panel\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — quantitative in vitro biophysical assays with mutagenesis panel; single lab but multiple orthogonal biophysical methods\",\n      \"pmids\": [\"16887812\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Mdm2 and MdmX prevent ASPP1/ASPP2 from stimulating the apoptotic function of p53 by binding and inhibiting the transcriptional activity of p53, without targeting p53 for degradation; both the DNA-binding and transactivation functions of p53 are required for ASPP1/ASPP2 stimulation.\",\n      \"method\": \"p53/mdm2 mutant panel; transcriptional reporter assays; co-immunoprecipitation; Western blot for p53 stability\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic mutant dissection plus reporter assays; single lab with two orthogonal approaches\",\n      \"pmids\": [\"15782125\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"53BP2/ASPP2 induces apoptosis via the mitochondrial death pathway: it localizes to mitochondria, causes depression of mitochondrial transmembrane potential (ΔΨm), and activates caspase-9 leading to PARP cleavage.\",\n      \"method\": \"Subcellular fractionation; mitochondrial membrane potential assay (JC-1); caspase-9 activity assay; annexin V staining; PARP cleavage Western blot\",\n      \"journal\": \"Genes to cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — subcellular fractionation localization plus functional mitochondrial assays; single lab\",\n      \"pmids\": [\"15743414\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"ASPP2 C-terminal ankyrin repeats and SH3 domain (ASPP2 Ank-SH3) mediate interaction with Bcl-2 family members (Bcl-2, Bcl-xL, Bcl-w) at two sites: the conserved BH4 motif and a proapoptotic regulator-binding site; within Bcl-2, binding to the BH4 domain is tightest; based on docking analysis ASPP2 is proposed to inhibit Bcl-2 function by occupying functional sites.\",\n      \"method\": \"Peptide array screening; surface plasmon resonance (SPR); isothermal titration calorimetry (ITC); computational docking; sequence alignment and peptide mutagenesis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — quantitative biophysical binding assays (SPR + ITC) plus peptide array plus structural modeling; multiple orthogonal methods in single rigorous study\",\n      \"pmids\": [\"18719108\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The C-termini of ASPP1 and ASPP2 directly bind the DNA-binding domains of p53, p63, and p73 with dissociation constants in the low micromolar range in a 1:1 stoichiometry; tri-complex formation between ASPPs, p53 family members, and PUMA/Bax DNA is mutually exclusive; uniquely, ASPP2 (but not ASPP1) forms a complex with PUMA and displaces p53 and p73.\",\n      \"method\": \"Surface plasmon resonance; isothermal titration calorimetry; EMSA (electrophoretic mobility shift assay); structure-based homology modelling\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — quantitative SPR and ITC plus EMSA; multiple orthogonal biochemical methods in one study\",\n      \"pmids\": [\"18676979\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The proline-rich domain of ASPP2 is natively unfolded and forms an intramolecular autoinhibitory interaction with its own Ank-SH3 domains, competing with intermolecular partner binding; ASPP2 Ank-SH3 (not the Pro domain) mediates interactions with partner-derived peptides; the presence of the Pro domain inhibits interactions mediated by Ank-SH3.\",\n      \"method\": \"CD spectroscopy; NMR; size exclusion chromatography; fluorescence anisotropy; peptide array screening; GST pull-down\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR and CD structural characterization plus multiple biochemical binding assays; multiple orthogonal methods in one study\",\n      \"pmids\": [\"18448430\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"ASPP2 binds Par-3 and controls its apical/junctional localization in neural progenitors; junctional localization of ASPP2 and Par-3 is interdependent; loss of ASPP2 in vivo disrupts tight/adherens junctions, impairs interkinetic nuclear migration, and causes neuroblastic rosette formation resembling primitive neuroepithelial tumors.\",\n      \"method\": \"In vivo mouse CNS development model (conditional knockdown/KO); co-immunoprecipitation; immunofluorescence localization; ASPP2 heterozygous and homozygous loss-of-function analysis\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP plus in vivo loss-of-function with specific morphological phenotypes; replicated across two independent papers (PMIDs 20619750 and 20619648)\",\n      \"pmids\": [\"20619750\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"ASPP2 interacts and colocalizes with PAR-3 at apical cell-cell junctions in polarized epithelial cells; depletion of ASPP2 causes polarity defects (tight junction formation, apical domain development) and mislocalization of PAR-3; disruption of the ASPP2–PAR-3 interaction causes the same polarity defects.\",\n      \"method\": \"Co-immunoprecipitation; immunofluorescence colocalization; siRNA depletion; domain-interaction mapping\",\n      \"journal\": \"Current biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP plus RNAi loss-of-function plus interaction-disruption mutants; independently replicated (PMID 20619750)\",\n      \"pmids\": [\"20619648\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"PP1A dephosphorylates TAZ at Ser-89 and Ser-311, promotes TAZ nuclear translocation, and stabilizes TAZ by disrupting SCF E3 ubiquitin ligase binding; ASPP2 facilitates the interaction between TAZ and PP1, thereby promoting TAZ dephosphorylation and TAZ-dependent gene expression.\",\n      \"method\": \"In vitro phosphatase assay; co-immunoprecipitation; phospho-specific antibody Western blot; nuclear/cytoplasmic fractionation; gene reporter assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro phosphatase assay plus Co-IP; single lab with two orthogonal methods\",\n      \"pmids\": [\"21189257\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"H. pylori CagA associates with ASPP2 upon translocation into host cells; this CagA–ASPP2 interaction recruits p53 into the complex, inhibits p53 apoptotic function, and leads to enhanced p53 degradation in an ASPP2-dependent manner; CagA-infected cells show increased resistance to doxorubicin-induced apoptosis requiring ASPP2.\",\n      \"method\": \"Co-immunoprecipitation from infected cells; apoptosis assay with doxorubicin; siRNA depletion of ASPP2; Western blot for p53 levels\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP from infected cells, ASPP2 RNAi epistasis, functional apoptosis readout; multiple orthogonal methods; independently replicated structurally (PMID 24474782)\",\n      \"pmids\": [\"21562218\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ASPP2 N-terminal RAS-association domain binds Ras-GTP at the plasma membrane and stimulates Ras-induced Raf/MEK/ERK signaling by promoting Ras-GTP loading, B-Raf/C-Raf dimerization, and C-Raf phosphorylation; decreased ASPP2 attenuates H-RasV12-induced senescence in normal human fibroblasts and keratinocytes; the short isoform BBP/53BP2S, lacking the N-terminus, fails to bind Ras-GTP or stimulate ERK.\",\n      \"method\": \"Ras-GTP pull-down; co-immunoprecipitation (Ras-GTP/ASPP2); pERK Western blot; plasma membrane colocalization by confocal microscopy; Raf dimerization assay; senescence assay (β-galactosidase); isoform comparison (53BP2S vs ASPP2)\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — GTP-Ras pull-down, dimerization assay, isoform-specific functional comparison, and in vivo senescence assay; multiple orthogonal methods\",\n      \"pmids\": [\"23248303\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ASPP1 and ASPP2 preferentially bind active (GTP-loaded) RAS via their N-terminal RAS-association domains; ASPP2 co-localizes with RAS at the cellular membrane and contributes to RAS membrane localization; in cancer cells, ASPP1/ASPP2 cooperate with oncogenic RAS to enhance p53 transcriptional apoptotic function.\",\n      \"method\": \"Pull-down with Ras-GTP (GTP-agarose); co-immunoprecipitation; confocal colocalization; luciferase reporter for p53 transcriptional targets; apoptosis assay\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Ras-GTP pull-down plus Co-IP plus colocalization; single lab corroborating PMID 23248303\",\n      \"pmids\": [\"23392125\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"FIH-1 (factor inhibiting HIF-1) hydroxylates ASPP2 at asparagine-986 within the ankyrin repeat domain; this hydroxylation is required for Par-3 binding to ASPP2—FIH-1 depletion impairs Par-3–ASPP2 interaction and causes relocation of ASPP2 from cell-cell contacts to the cytosol—without affecting p53 binding, apoptosis, or proliferation.\",\n      \"method\": \"Mass spectrometry identification of hydroxylation site; site-directed mutagenesis; co-immunoprecipitation; immunofluorescence localization; siRNA depletion of FIH-1; FIH-1 inhibitor (DMOG)\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — MS identification of PTM site, mutagenesis confirmation, and Co-IP-based functional consequence; multiple orthogonal methods in one study\",\n      \"pmids\": [\"23606740\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ASPP2 inhibits ΔNp63 expression through its ability to bind IκB and enhance nuclear RelA/p65 (NF-κB), which mediates transcriptional repression of p63; heterozygosity of p63 (but not p53) prevents squamous cell carcinoma development in ASPP2-haploinsufficient mice.\",\n      \"method\": \"Co-immunoprecipitation (ASPP2–IκB interaction); nuclear fractionation; ChIP for RelA/p65 on p63 promoter; genetic epistasis (p63+/−; p53+/− crosses with ASPP2Δexon3/+ mice); tumor incidence analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP, ChIP, and genetic epistasis in mouse model; multiple orthogonal methods establishing pathway position\",\n      \"pmids\": [\"24127607\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ASPP2 intramolecular autoinhibitory interaction: the disordered proline-rich domain of ASPP2 competes with p53 core domain for binding the n-src loop of the ASPP2 SH3 domain; p53 core domain and NFκB (residues 303–332) bind partially overlapping sites on the ASPP2 SH3 RT loop; Bcl-2 binds ASPP2 at sites largely distinct from p53/NFκB.\",\n      \"method\": \"Fluorescence anisotropy competition experiments; peptide-based binding studies; recombinant protein assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — quantitative biophysical competition experiments with recombinant proteins; single lab extending prior structural work\",\n      \"pmids\": [\"23472201\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ASPP2 is a novel substrate of MAPK (ERK); MAPK phosphorylation of ASPP2 is required for RAS-induced increased binding to p53 and enhanced transactivation of pro-apoptotic genes; a phosphorylation-deficient ASPP2 mutant shows reduced p53 binding and fails to enhance apoptosis.\",\n      \"method\": \"In vitro MAPK kinase assay; phosphorylation-deficient ASPP2 mutant; co-immunoprecipitation of ASPP2–p53; luciferase reporter assay; apoptosis assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay plus mutagenesis plus functional Co-IP; single lab\",\n      \"pmids\": [\"24312625\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ASPP2 attenuates Src kinase activation in a Csk (C-terminal Src kinase)-dependent manner; ASPP2 (but not ASPP1) transfection decreases Src-pY416 phosphorylation; this ASPP2-mediated Src inactivation reduces cell migration.\",\n      \"method\": \"Transfection of ASPP2 vs ASPP1; Western blot for Src-pY416; Csk siRNA epistasis; wound-healing/migration assay\",\n      \"journal\": \"Carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Csk RNAi epistasis plus specific phospho-readout; single lab, two methods\",\n      \"pmids\": [\"23671128\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"DDA3 (a p53 target oncoprotein) binds ASPP2 via its residues 118–241 to both N- and C-terminal regions of ASPP2; DDA3 dose-dependently inhibits ASPP2-stimulated p53-mediated BAX promoter activation without interfering with ASPP2–p53 binding.\",\n      \"method\": \"Yeast two-hybrid screen; GST pull-down; immunofluorescence colocalization; domain mapping; luciferase BAX promoter reporter assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — GST pull-down confirmed by reporter functional assay; single lab\",\n      \"pmids\": [\"18793611\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Crystal structure (2.0 Å) of the N-terminal CagA subdomain bound to a 7-kDa proline-rich sequence of ASPP2: CagA forms a three-helix bundle with a deep binding cleft for a 20-aa conserved ASPP2 peptide that adopts an extended helix; structure-based loss-of-contact mutations in either CagA or ASPP2 disrupt the interaction in vitro and in vivo and alter ASPP2 function.\",\n      \"method\": \"X-ray crystallography (2.0 Å co-crystal); yeast two-hybrid domain delineation; in vitro biochemical binding confirmation; structure-based mutagenesis; functional cell-based assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — atomic resolution co-crystal structure with mutagenesis validation in vitro and in vivo\",\n      \"pmids\": [\"24474782\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ASPP2 induces mesenchymal-to-epithelial transition (MET) via its PAR3-binding N-terminus (independently of p53 binding); mechanistically, ASPP2 prevents β-catenin from transactivating ZEB1 by (i) forming an ASPP2–β-catenin–E-cadherin ternary complex and (ii) inhibiting N-terminal phosphorylation of β-catenin to stabilize the β-catenin–E-cadherin complex; ASPP2 limits oncogenic RAS pro-invasive effects and inhibits tumor metastasis in vivo.\",\n      \"method\": \"Co-immunoprecipitation (ASPP2–β-catenin–E-cadherin ternary complex); domain-mapping (N-terminus vs. p53-binding domain); β-catenin phosphorylation assay; in vivo mouse kidney MET model; in vivo metastasis assay\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — ternary complex Co-IP, domain-specific rescue, in vivo models; multiple orthogonal methods\",\n      \"pmids\": [\"25344754\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ASPP2 forms an apical-lateral polarity complex at tight junctions acting as a scaffold for PP1 and junctional YAP via dedicated binding domains; ASPP2 directly induces dephosphorylation and activation of junctional YAP; this mechanism controls YAP function in polarized epithelial cells and in the murine colonic epithelium in vivo.\",\n      \"method\": \"Co-immunoprecipitation (ASPP2–PP1–YAP complex); domain mapping; phospho-YAP Western blot; siRNA depletion; in vivo murine colonic epithelium analysis\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP scaffold complex plus phosphorylation assay plus in vivo; single lab\",\n      \"pmids\": [\"25360797\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ASPP2 induces autophagic apoptosis in hepatoma cells through p53/p73-independent CHOP expression; CHOP decreases Bcl-2 expression, releasing Beclin-1 from Bcl-2–Beclin-1 complexes to initiate autophagy; ASPP2 also induces DRAM expression; CHOP promotes nuclear translocation of Bcl-2 where it is sequestered in ASPP2–Bcl-2 nuclear complexes, preventing Bcl-2 return to the cytoplasm.\",\n      \"method\": \"Western blot; co-immunoprecipitation (ASPP2–Bcl-2); siRNA knockdown of CHOP; adenoviral ASPP2 overexpression; autophagy and apoptosis assays; nuclear/cytoplasmic fractionation\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus RNAi pathway dissection plus compartment fractionation; single lab\",\n      \"pmids\": [\"25032846\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The E3 ubiquitin ligase Itch mediates ASPP2 degradation and ubiquitination via interaction of the ASPP2 PPXY motif with Itch WW domains; Yap1 competes with Itch for binding to ASPP2 and prevents Itch-mediated ASPP2 degradation, indicating antagonistic regulation of ASPP2 protein stability.\",\n      \"method\": \"Co-immunoprecipitation; ubiquitination assay in vivo; domain mapping (PPXY–WW interaction); competition binding assay; protein stability (CHX chase)\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ubiquitination assay plus Co-IP competition; single lab\",\n      \"pmids\": [\"25436413\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The ubiquitin E3 ligase Siah2 interacts with ASPP2 and ASPP1 under hypoxic conditions and targets ASPP2 for ubiquitination and proteasomal degradation via degron motifs in ASPP2; Siah2 inhibition increases ASPP2 levels and enhances tight junction integrity and polarity in 3D organotypic culture; hypoxia-induced Siah2 upregulation decreases ASPP2 levels and impairs polarity.\",\n      \"method\": \"LC-MS/MS identification of Siah2–ASPP2 interaction; co-immunoprecipitation; ubiquitination assay; degron mutant mapping; 3D organotypic culture; siRNA depletion; polarity assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-based interaction identification confirmed by Co-IP and ubiquitination assay with functional polarity readout; single lab\",\n      \"pmids\": [\"23644657\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"ASPP2 physically interacts with C-terminal Src kinase (CSK) and stimulates its kinase activity, leading to Src inactivation, AP1-mediated downregulation of Snail expression, and suppression of HCC stemness; pharmacological inhibition of Src attenuates ASPP2-deficiency effects.\",\n      \"method\": \"Co-immunoprecipitation (ASPP2–CSK); CSK kinase activity assay; Src phosphorylation Western blot; AP1/Snail reporter; tumor sphere formation; side-population assay; Src inhibitor rescue\",\n      \"journal\": \"Tumour biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus kinase activity assay plus pharmacological rescue; single lab\",\n      \"pmids\": [\"27473084\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"An intrinsically disordered region of ASPP2 (residues 448–692) that is unstructured in solution binds the N-terminal domain of CagA; peptide array mapping identified multiple distributed interaction sites throughout ASPP2 for CagA, extending beyond the crystallographically resolved fragment.\",\n      \"method\": \"SEC-MALS; circular dichroism; NMR; surface plasmon resonance; isothermal titration calorimetry; peptide array mapping\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — multiple biophysical methods (NMR, ITC, SPR, CD) in one rigorous study\",\n      \"pmids\": [\"25963096\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"ASPP1 and ASPP2 interact with centrosome linker protein C-Nap1; co-depletion of ASPP1/2 inhibits C-Nap1 re-association with centrosomes at mitotic exit and impairs centrosome linker reassembly; ASPP1/2 facilitate PP1α–C-Nap1 interaction and antagonize NEK2A-mediated C-Nap1 Ser2417/2421 phosphorylation in a PP1-dependent manner.\",\n      \"method\": \"Co-immunoprecipitation (ASPP2–C-Nap1, ASPP2–PP1α); siRNA co-depletion; phospho-C-Nap1 (Ser2417/2421) Western blot; centrosome immunofluorescence; NEK2A kinase assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus RNAi epistasis plus phospho-readout; single lab\",\n      \"pmids\": [\"25660448\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Crystal structure of the p73 DNA-binding domain (DBD) bound to ASPP2 ankyrin repeat and SH3 domains solved at high resolution; p73 DBD exhibits a divergent L2 loop (two-residue insertion that repacks the p53 R175 hotspot equivalent); ASPP2 binding is preserved via conformational adaptation in both the ankyrin repeat and SH3 domains.\",\n      \"method\": \"X-ray crystallography (high-resolution crystal structures of free p73 DBD and p73 DBD–ASPP2 Ank-SH3 complex)\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — atomic-resolution crystal structure; single lab but high-quality structural data\",\n      \"pmids\": [\"22917970\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ASPP2 suppresses TGF-β1-induced EMT in gastric cancer cells by interacting with E3 ubiquitin ligase ITCH and inhibiting ITCH-mediated degradation of Smad7 (a negative regulator of TGF-β1–Smad2/3 signaling); ASPP2 promotes PAR3 recruitment to cell-cell junctions.\",\n      \"method\": \"Co-immunoprecipitation (ASPP2–ITCH); Smad7 degradation assay; TGF-β1–Smad2/3 signaling Western blot; immunofluorescence (PAR3 junctional localization); migration/invasion assay; in vivo peritoneal dissemination model\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus functional degradation assay plus in vivo model; single lab\",\n      \"pmids\": [\"28400336\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"ASPP2 interacts with SREBP-2 in the nucleus and restricts SREBP-2 transcriptional activity on mevalonate pathway target genes (including HMGCR); ASPP2 depletion increases cholesterol levels and tumor-initiating capability; simvastatin rescues ASPP2-depletion-induced phenotypes.\",\n      \"method\": \"Co-immunoprecipitation (ASPP2–SREBP-2 nuclear); ChIP/reporter assay for SREBP-2 targets; cholesterol measurement; gene expression profiling; xenograft model; simvastatin pharmacological rescue\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — nuclear Co-IP plus gene expression profiling plus pharmacological rescue; single lab\",\n      \"pmids\": [\"31685796\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ASPP2 inhibits NF-κB–induced transcription of BECN1 (Beclin-1) directly via an ASPP2–p65/RelA–IκBα complex that inhibits IκBα phosphorylation and p65 nuclear translocation; ASPP2 also binds BECN1 and reshapes the PIK3C3 complex (decreasing PIK3C3–UVRAG interaction, increasing Rubicon binding), thereby inhibiting autophagy initiation.\",\n      \"method\": \"Co-immunoprecipitation (ASPP2–p65–IκBα complex; ASPP2–BECN1); IκBα phosphorylation assay; BECN1 promoter-reporter assay; PIK3C3 complex pull-down; autophagic flux assay; xenograft model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple Co-IPs plus promoter assay plus complex reshaping; single lab\",\n      \"pmids\": [\"27929538\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CagA–ASPP2 interaction promotes remodeling of the PAR polarity complex and loss of cell polarity in H. pylori-infected gastric epithelial cells; blockade of ASPP2 signaling by EGFR inhibitors or a CagA-binding ASPP2 peptide prevents polarity loss and decreases H. pylori survival in infected gastric organoids.\",\n      \"method\": \"Gastric organoid infection model; high-content imaging screen; co-immunoprecipitation/proximity ligation; EGFR inhibitor treatment; synthetic ASPP2 decoy peptide\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — organoid functional model plus pharmacological and peptide rescue; single lab\",\n      \"pmids\": [\"31964836\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Truncated ASPP2 (t-ASPP2, N-terminal truncation) induces actomyosin relaxation via interaction with PP1 to enable survival of E-cadherin-deficient mammary epithelial cells on stiff matrices (required for ILC initiation); separately, t-ASPP2-induced YAP activation (not actomyosin relaxation) contributes to tumor growth and progression.\",\n      \"method\": \"Mouse ILC model with E-cadherin loss; actomyosin tension assay; PP1 interaction domain mapping; YAP activity assay; conditional mouse genetics (PTEN loss)\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetically engineered mouse model with mechanistic domain mapping and pathway dissection; single lab\",\n      \"pmids\": [\"32060147\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"An alternatively spliced ASPP2 isoform, ASPP2κ (exon skipping generating truncated C-terminus lacking p53-binding sites), acts as a dominant-negative variant impairing TP53-dependent apoptosis induction; ASPP2κ expression causes perturbed proliferation, impaired apoptosis, mitotic failure, and chromosomal instability; its expression is stress-inducible.\",\n      \"method\": \"mRNA splice variant screening; isoform-specific PCR and epitope-specific antibody generation; forced expression and isoform-specific RNAi in cell models; apoptosis, proliferation, and chromosomal instability assays\",\n      \"journal\": \"EBioMedicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — isoform-specific KD/OE with defined molecular and cellular phenotypes; single lab\",\n      \"pmids\": [\"30952616\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ASPP2 binds HSF1 in the cytoplasm of HBV-infected cells, preventing HSF1 nuclear translocation and thereby inhibiting transcriptional activation of ATG7; by suppressing ATG7 expression, ASPP2 reduces HBV-induced hepatocyte autophagy and inhibits HBV replication.\",\n      \"method\": \"Co-immunoprecipitation (ASPP2–HSF1); nuclear/cytoplasmic fractionation; ChIP for HSF1 on ATG7 promoter; Western blot for ATG7; autophagic flux assay; HBV replication assay\",\n      \"journal\": \"Journal of cellular and molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus nuclear fractionation plus ChIP; single lab with multiple methods\",\n      \"pmids\": [\"34085409\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ATF4 transcription factor binds the TP53BP2 promoter (ChIP-validated) and drives TP53BP2 expression during ischemia/reperfusion stress; TP53BP2 overexpression promotes inflammation and apoptosis in hepatocytes, and sufentanil protection is mediated by suppression of ATF4-driven TP53BP2 induction.\",\n      \"method\": \"ChIP assay (ATF4 on TP53BP2 promoter); TP53BP2 overexpression in H/R cell model; Western blot; ELISA for inflammatory markers; in vivo rat I/R model\",\n      \"journal\": \"Inflammation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP identification of transcriptional regulator plus functional overexpression assay; single lab\",\n      \"pmids\": [\"33751357\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"DEAD-box protein Ddx42p physically interacts with ASPP2 via the Ddx42p C-terminal region and ASPP2 mid-N-terminal + Ank-SH3 domains; Ddx42p overexpression interferes with ASPP2-induced apoptosis; elevated Ddx42p shifts ASPP2 localization from nucleus+cytoplasm to predominantly cytoplasm.\",\n      \"method\": \"Co-immunoprecipitation; domain mapping; apoptosis assay with Ddx42p overexpression/knockdown; immunofluorescence localization\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP with localization; single lab, limited validation\",\n      \"pmids\": [\"19377511\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TP53BP2 downregulates SOCS2 expression, thereby facilitating JAK/STAT signaling and enhancing the anti-HBV effect of IFN-α in hepatocytes.\",\n      \"method\": \"In vitro and in vivo experiments; SOCS2 expression measurement upon TP53BP2 loss/gain; JAK/STAT pathway readouts; antiviral assay with IFN-α\",\n      \"journal\": \"Journal of hepatology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — functional pathway assay in single study without detailed mechanistic dissection of direct interaction\",\n      \"pmids\": [\"37858684\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ASPP2 (TP53BP2) is a multifunctional scaffold protein whose C-terminal ankyrin repeats and SH3 domain directly bind the p53 (and p63/p73) DNA-binding domain, selectively stimulating transcription of pro-apoptotic target genes (Bax, PUMA, PIG3) over cell-cycle arrest genes; this interaction is regulated by an intramolecular autoinhibitory mechanism involving the natively disordered N-terminal proline-rich domain, by MAPK phosphorylation (which enhances p53 binding), by proteasomal degradation (controlled by the E3 ligases Itch and Siah2), and by FIH-1-mediated hydroxylation of Asn-986; the same Ank-SH3 domain also binds Bcl-2 (via the BH4 domain), NF-κB p65/RelA (in an IκB-like manner), and APP-BP1; independently of p53, ASPP2 regulates epithelial polarity by binding PAR-3 (interaction dependent on FIH-1 hydroxylation) and acting as a PP1 scaffold that dephosphorylates and activates junctional YAP/TAZ; its N-terminal RAS-association domain binds active Ras-GTP, potentiates Raf/MEK/ERK signaling to promote oncogene-induced senescence, and is required for cooperation with oncogenic RAS to enhance p53-dependent apoptosis; ASPP2 also suppresses the mevalonate pathway by binding nuclear SREBP-2, inhibits autophagy by reshaping the PIK3C3–Beclin-1 complex and by sequestering HSF1 to prevent ATG7 transcription, and represses p63 expression via NF-κB–mediated transcriptional suppression; the H. pylori oncoprotein CagA exploits the ASPP2 proline-rich domain to form a high-affinity complex that subverts ASPP2 polarity and apoptosis functions.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ASPP2 (TP53BP2) is a multidomain scaffold protein that couples the p53 family to programmed cell death and links cell polarity, RAS signaling, and metabolic control to tumor suppression [#0, #5, #21]. Its C-terminal ankyrin repeats and SH3 domain dock onto the DNA-binding domains of p53, p63, and p73 — engaging the L2 and L3 loops of the p53 core in a mode that overlaps the DNA-binding surface, such that the six most frequent cancer-associated p53 mutations disrupt the interaction and ASPP2 binding is mutually exclusive with response-element DNA binding [#0, #11, #15, #38]. Through this interface ASPP2 selectively stimulates transcription of pro-apoptotic targets (Bax, PUMA, PIG3) rather than cell-cycle arrest genes, and much of its p53-independent apoptotic output is relayed through p63/p73 [#5]. The same Ank-SH3 module binds Bcl-2 family proteins at the conserved BH4 motif and engages NF-\\u03baB p65/RelA, while a natively disordered N-terminal proline-rich domain folds back to autoinhibit Ank-SH3, gating partner access [#1, #14, #16, #25]. ASPP2 activity is tuned by MAPK/ERK phosphorylation that enhances p53 binding upon RAS activation, by FIH-1 hydroxylation of Asn-986 that licenses Par-3 binding, and by proteasomal turnover through the E3 ligases Itch and Siah2 [#26, #23, #33, #34]. Beyond apoptosis, ASPP2 acts at apical cell-cell junctions: it binds Par-3 to maintain epithelial and neuroepithelial polarity and scaffolds PP1 to dephosphorylate and activate junctional YAP/TAZ [#17, #18, #19, #31]. Its N-terminal RAS-association domain binds Ras-GTP at the membrane, potentiating Raf/MEK/ERK signaling and oncogene-induced senescence and cooperating with oncogenic RAS to enhance p53-dependent apoptosis [#21, #22]. ASPP2 further suppresses tumorigenic programs by restraining \\u0394Np63 via NF-\\u03baB, blocking \\u03b2-catenin-driven ZEB1 to promote mesenchymal-to-epithelial transition, inhibiting autophagy initiation, and restricting nuclear SREBP-2 control of the mevalonate pathway [#24, #30, #41, #40]. The H. pylori oncoprotein CagA exploits the disordered ASPP2 proline-rich region to form a high-affinity complex that subverts ASPP2 polarity and apoptotic functions [#20, #29, #42].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Established the structural basis for how ASPP2 recognizes p53 and why the interaction is tumor-relevant, answering how a non-DNA-binding partner engages the p53 core.\",\n      \"evidence\": \"X-ray crystallography of the p53 core\\u201353BP2 complex with cancer-mutant in vitro binding assays\",\n      \"pmids\": [\"8875926\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not show whether binding occurs on or off DNA in cells\", \"No functional transcriptional consequence quantified\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Showed ASPP2 also binds Bcl-2 through the same Ank-SH3 domains and that Bcl-2 and p53 compete, raising the possibility of partner-switching control of apoptosis.\",\n      \"evidence\": \"Yeast two-hybrid, in vitro GST pull-down with competition assays, immunofluorescence, flow cytometry\",\n      \"pmids\": [\"8668206\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological relevance of competition not established in vivo\", \"Cytoplasmic colocalization only partial\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Resolved the puzzle that cytoplasmic ASPP2 still enhances p53 transcriptional output, framing it as a signal-transduction modulator rather than an obligatory nuclear cofactor.\",\n      \"evidence\": \"Immunofluorescence localization plus p53-dependent reporter and target-protein assays\",\n      \"pmids\": [\"9748285\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism connecting cytoplasmic ASPP2 to nuclear p53 unresolved\", \"Single-lab observation\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Identified NF-\\u03baB p65/RelA as a direct ASPP2 partner that suppresses ASPP2-induced apoptosis and mapped apoptotic activity to the full-length protein.\",\n      \"evidence\": \"Yeast two-hybrid, in vitro and mammalian two-hybrid, GFP localization, transfection apoptosis assay\",\n      \"pmids\": [\"10498867\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Domain of p65 binding not precisely mapped here\", \"p53-dependence of the apoptotic effect not isolated\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Placed ASPP2 in the DNA-damage response by showing UV induces its protein in a p53-independent manner while wild-type p53 suppresses it, defining a feedback relationship that lowers the apoptotic threshold.\",\n      \"evidence\": \"Western blot in defined-genotype lines, inducible expression, antisense knockdown, clonogenic and apoptosis assays\",\n      \"pmids\": [\"11027272\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of p53-mediated ASPP2 suppression not defined\", \"Post-transcriptional vs transcriptional control unclear\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Defined the central functional principle: ASPPs selectively drive apoptotic over arrest p53 targets and extend this to p63/p73, with p63/p73 carrying p53-independent apoptosis.\",\n      \"evidence\": \"Co-IP, in vitro binding, promoter-selective luciferase reporters, p63/p73 RNAi epistasis, apoptosis assays\",\n      \"pmids\": [\"14729977\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of promoter selectivity not structurally explained\", \"How ASPP2 distinguishes apoptotic from arrest promoters unknown\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Connected ASPP2 to neddylation control by showing it binds APP-BP1 and inhibits NEDD8 conjugation to Cullin-1, with consequences for proliferation and neuronal apoptosis.\",\n      \"evidence\": \"Endogenous Co-IP, domain mapping, NEDD8 conjugation assay, proliferation and apoptosis assays\",\n      \"pmids\": [\"12694406\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Integration with p53/apoptotic functions unclear\", \"Single-lab finding\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Defined gene architecture (53BP2S short and 53BP2L/ASPP2 long isoforms) by alternative splicing, providing the basis for isoform-specific functions.\",\n      \"evidence\": \"RT-PCR, genomic cloning, sequencing of TP53BP2 transcripts\",\n      \"pmids\": [\"14766226\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional differences between isoforms not assessed here\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Showed HCV core protein competes with p53 for ASPP2, an early example of viral subversion of ASPP2-enhanced apoptosis.\",\n      \"evidence\": \"Yeast two-hybrid, in vitro competition binding, apoptosis and reporter assays\",\n      \"pmids\": [\"14985081\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Binding site on ASPP2 not mapped\", \"In-cell relevance during infection not tested\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Multiple studies defined ASPP2 regulatory inputs and outputs: E2F transcriptional induction in S-phase, proteasomal stability control, the mutually exclusive p53/DNA-binding mechanism, Mdm2/MdmX antagonism, and a mitochondrial apoptotic pathway.\",\n      \"evidence\": \"ChIP and promoter reporters; proteasome inhibition and ubiquitination assays; biophysical binding (SPR/ITC/anisotropy) with p53 mutant panels; mutant dissection; subcellular fractionation with caspase-9 assays\",\n      \"pmids\": [\"15592436\", \"15731768\", \"16091363\", \"16887812\", \"15782125\", \"15743414\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"E3 ligase mediating ASPP2 ubiquitination not yet identified in this period\", \"How cytoplasmic/mitochondrial ASPP2 ties to nuclear p53 transcription unresolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Quantitative biophysics and NMR established the autoinhibitory architecture and binding thermodynamics: the disordered Pro domain competes with Ank-SH3 for partner peptides, and ASPP2 binds Bcl-2 family BH4 motifs and p53/p63/p73 DBDs at low-micromolar 1:1 stoichiometry, with ASPP2 uniquely binding PUMA DNA.\",\n      \"evidence\": \"CD, NMR, SEC, fluorescence anisotropy, peptide arrays, SPR, ITC, EMSA, homology modeling\",\n      \"pmids\": [\"18719108\", \"18676979\", \"18448430\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Trigger that relieves intramolecular autoinhibition in cells not defined\", \"Functional impact of PUMA-DNA binding on transcription unresolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identified DDA3 as a p53-target oncoprotein that binds ASPP2 and inhibits its stimulation of p53-mediated BAX activation without blocking ASPP2\\u2013p53 binding.\",\n      \"evidence\": \"Yeast two-hybrid, GST pull-down, colocalization, domain mapping, BAX reporter assay\",\n      \"pmids\": [\"18793611\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of inhibition without disrupting p53 binding unclear\", \"Single-lab finding\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Revealed a p53-independent role in epithelial/neuroepithelial polarity by showing ASPP2 binds Par-3 and maintains junctions, with loss causing neuroepithelial tumor-like phenotypes in vivo.\",\n      \"evidence\": \"In vivo mouse CNS loss-of-function, Co-IP, immunofluorescence\",\n      \"pmids\": [\"20619750\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Biochemical determinants of the Par-3 interaction not mapped here\", \"Link between polarity loss and tumorigenesis mechanistic at this stage\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Extended the polarity role to epithelial cells and mechanistically linked ASPP2 to YAP/TAZ regulation, showing it scaffolds PP1 to dephosphorylate TAZ and facilitates the Par-3 apical complex.\",\n      \"evidence\": \"Co-IP, immunofluorescence, siRNA depletion, in vitro phosphatase assay, fractionation, reporter assays\",\n      \"pmids\": [\"20619648\", \"21189257\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How polarity and apoptotic functions are coordinated unclear\", \"PP1 scaffolding stoichiometry not defined\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Defined the N-terminal RAS-association function: ASPP2 binds Ras-GTP at the membrane, potentiates Raf/MEK/ERK signaling and oncogene-induced senescence, distinguishing the long isoform from senescence-incompetent 53BP2S.\",\n      \"evidence\": \"Ras-GTP pull-down, Co-IP, pERK Western, confocal colocalization, Raf dimerization, senescence assay, isoform comparison\",\n      \"pmids\": [\"23248303\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How RAS engagement feeds back to p53/apoptosis not yet linked here\", \"Structural basis of RA-domain\\u2013Ras binding not solved\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Crystallography of the p73 DBD\\u2013ASPP2 Ank-SH3 complex showed conformational adaptation accommodates the divergent p73 L2 loop, explaining conserved binding across the p53 family.\",\n      \"evidence\": \"X-ray crystallography of free and ASPP2-bound p73 DBD\",\n      \"pmids\": [\"22917970\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Affinity differences across family members not functionally resolved here\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"A cluster of studies defined ASPP2 post-translational and signaling regulation: FIH-1 hydroxylation of Asn-986 gating Par-3 binding, MAPK phosphorylation enhancing p53 binding upon RAS activation, NF-\\u03baB-mediated \\u0394Np63 repression as a tumor-suppressive axis, mapped competing autoinhibitory/partner sites on the SH3, Csk-dependent Src inactivation, Siah2-mediated hypoxic degradation, and RAS cooperation to enhance p53 apoptosis.\",\n      \"evidence\": \"MS PTM mapping with mutagenesis and Co-IP; in vitro kinase assays with phospho-mutants; Co-IP/ChIP and mouse genetic epistasis; biophysical competition; Csk RNAi epistasis with migration; LC-MS/MS, ubiquitination and 3D polarity assays; Ras-GTP pull-down with apoptosis reporters\",\n      \"pmids\": [\"23606740\", \"24312625\", \"24127607\", \"23472201\", \"23671128\", \"23644657\", \"23392125\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Crosstalk between hydroxylation, phosphorylation, and degradation inputs not integrated\", \"Stimulus-specific deployment of distinct ASPP2 functions unresolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Defined the CagA\\u2013ASPP2 hijacking mechanism structurally and functionally, and expanded ASPP2 tumor-suppressive functions to MET, YAP/PP1 junctional scaffolding, autophagic apoptosis via CHOP-Bcl-2-Beclin-1, and Itch-mediated degradation antagonized by Yap1.\",\n      \"evidence\": \"2.0 \\u00c5 co-crystal with mutagenesis; Co-IP of ternary \\u03b2-catenin/E-cadherin complex with in vivo MET/metastasis models; PP1\\u2013YAP scaffold Co-IP with in vivo colonic epithelium; CHOP RNAi pathway dissection; PPXY\\u2013WW ubiquitination and competition assays\",\n      \"pmids\": [\"24474782\", \"25344754\", \"25360797\", \"25032846\", \"25436413\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Hierarchy among ASPP2 polarity, MET, and apoptotic outputs unclear\", \"Whether these functions operate in the same cell context not established\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Refined CagA binding to a distributed disordered ASPP2 region, defined ASPP2\\u2013Csk\\u2013Src\\u2013Snail suppression of HCC stemness, and identified an ASPP1/2\\u2013PP1\\u03b1\\u2013C-Nap1 axis in centrosome linker reassembly.\",\n      \"evidence\": \"SEC-MALS/NMR/SPR/ITC/peptide arrays; Co-IP with Csk kinase and AP1/Snail reporters and stemness assays; Co-IP with phospho-C-Nap1 and centrosome imaging\",\n      \"pmids\": [\"25963096\", \"27473084\", \"25660448\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mitotic centrosome role not connected to apoptotic/polarity functions\", \"Csk-stimulating mechanism not structurally defined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showed ASPP2 suppresses TGF-\\u03b21-induced EMT by stabilizing Smad7 through inhibition of ITCH-mediated degradation, integrating ASPP2 into junctional and TGF-\\u03b2 anti-tumor control.\",\n      \"evidence\": \"Co-IP, Smad7 degradation assay, Smad2/3 Western, immunofluorescence, migration/invasion and in vivo dissemination\",\n      \"pmids\": [\"28400336\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relationship between ASPP2\\u2013ITCH stabilization of Smad7 and Itch-mediated ASPP2 degradation unclear\", \"Single-lab finding\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Linked ASPP2 to lipid metabolism by showing it restrains nuclear SREBP-2 control of the mevalonate pathway, and identified a dominant-negative splice isoform (ASPP2\\u03ba) that impairs p53 apoptosis and drives chromosomal instability.\",\n      \"evidence\": \"Nuclear Co-IP, ChIP/reporter, cholesterol measurement, xenograft and simvastatin rescue; isoform-specific KD/OE with CIN assays\",\n      \"pmids\": [\"31685796\", \"30952616\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs indirect SREBP-2 inhibition mechanism not fully resolved\", \"Regulation of ASPP2\\u03ba splicing under stress not detailed\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Defined CagA-driven PAR-complex remodeling in gastric organoids and a truncated ASPP2\\u2013PP1 actomyosin/YAP mechanism enabling E-cadherin-deficient epithelial survival in lobular carcinoma.\",\n      \"evidence\": \"Organoid infection with high-content imaging, EGFR-inhibitor and decoy-peptide rescue; mouse ILC model with PP1 domain mapping and YAP dissection\",\n      \"pmids\": [\"31964836\", \"32060147\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Generality of t-ASPP2 mechanism beyond ILC unclear\", \"How truncation alters ASPP2 partner selection not detailed\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Established autophagy/transcription control roles in viral and stress contexts: ASPP2 sequesters HSF1 to block ATG7 transcription and limit HBV-induced autophagy, and is itself an ATF4 transcriptional target driving inflammation and apoptosis during ischemia/reperfusion.\",\n      \"evidence\": \"Co-IP, fractionation and ChIP for HSF1\\u2013ATG7; ChIP of ATF4 on TP53BP2 promoter with overexpression and in vivo I/R models\",\n      \"pmids\": [\"34085409\", \"33751357\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ASPP2 contact residues on HSF1 not mapped\", \"Integration with NF-\\u03baB/Beclin-1 autophagy control unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Defined dual autophagy suppression: ASPP2 inhibits NF-\\u03baB-driven BECN1 transcription via an ASPP2\\u2013p65\\u2013I\\u03baB\\u03b1 complex and reshapes the PIK3C3\\u2013Beclin-1 complex to block autophagy initiation.\",\n      \"evidence\": \"Co-IP, I\\u03baB\\u03b1 phosphorylation assay, BECN1 promoter reporter, PIK3C3 complex pull-down, autophagic flux, xenograft\",\n      \"pmids\": [\"27929538\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Balance between pro-autophagic (CHOP axis) and anti-autophagic ASPP2 effects unresolved\", \"Single-lab finding\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How ASPP2's distinct modular functions \\u2014 p53-family apoptosis, RAS signaling, junctional polarity/YAP, autophagy and metabolic control \\u2014 are coordinated and selectively deployed within a single cell remains unresolved.\",\n      \"evidence\": \"No single study integrates the autoinhibition, PTM, and partner-switching inputs into a unified regulatory logic\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of full-length ASPP2 with multiple partners\", \"Stimulus-to-function mapping incomplete\", \"Role of low-confidence partners (Ddx42p, SOCS2 axis) not validated\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [17, 18, 19, 31, 37, 21]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [5, 11, 14, 16, 6]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [5, 24, 40]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [15]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2, 3, 23, 45]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [40, 47, 32]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [21, 22, 17, 18]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [13]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [37]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [0, 5, 13, 4]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [21, 22, 26, 19]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [5, 24, 40]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [41, 32, 45]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [10, 33, 34, 6]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [17, 18, 30]}\n    ],\n    \"complexes\": [\"ASPP2\\u2013PP1\\u2013YAP junctional polarity complex\", \"ASPP2\\u2013p65/RelA\\u2013I\\u03baB\\u03b1 complex\", \"ASPP2\\u2013\\u03b2-catenin\\u2013E-cadherin complex\", \"PIK3C3\\u2013Beclin-1 complex\"],\n    \"partners\": [\"TP53\", \"TP73\", \"BCL2\", \"RELA\", \"PARD3\", \"RAS\", \"PPP1CA\", \"ITCH\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":9,"faith_total":9,"faith_pct":100.0}}