{"gene":"CCAR2","run_date":"2026-06-09T22:57:17","timeline":{"discoveries":[{"year":2007,"finding":"DBC-1/CCAR2 amino terminus binds directly to the ERα hormone-binding domain in a ligand-independent manner, stabilizing unliganded ERα protein; antiestrogens (tamoxifen, ICI 182,780) and estrogen disrupt this interaction. RNAi-mediated DBC-1 depletion reduces unliganded ERα protein levels and promotes hormone-independent apoptosis of ERα-positive breast cancer cells.","method":"In vitro binding assay, co-immunoprecipitation, RNA interference (siRNA knockdown), apoptosis assays","journal":"Molecular endocrinology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — direct in vitro binding plus in vivo co-IP, RNAi functional readout with specific phenotype, single lab but multiple orthogonal methods","pmids":["17473282"],"is_preprint":false},{"year":2013,"finding":"hMOF (MYST-family acetyltransferase) acetylates DBC1/CCAR2 on lysine residues K112 and K215; this acetylation inhibits DBC1 binding to SIRT1, thereby increasing SIRT1 deacetylase activity. SIRT1 in turn promotes DBC1 deacetylation, forming a negative-feedback loop. After DNA damage, ATM-dependent inhibition of hMOF binding to DBC1 reduces DBC1 acetylation and enhances DBC1–SIRT1 binding. A DBC1 acetylation-mimic mutant fails to promote apoptosis after DNA damage.","method":"Mass spectrometry, co-immunoprecipitation, in vitro acetyltransferase assay, site-directed mutagenesis, deacetylase activity assay, apoptosis assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro enzymatic assay, MS identification of sites, mutagenesis, reciprocal Co-IP, functional apoptosis readout in single rigorous study","pmids":["24126058"],"is_preprint":false},{"year":2015,"finding":"CCAR2 is required for Chk2-dependent phosphorylation of KAP1, a step needed for heterochromatin relaxation and repair of heterochromatic DNA double-strand breaks. CCAR2 knockout cells exhibit defective Chk2 activation, elevated KAP1 non-phosphorylation, and persistent DNA damage foci specifically in heterochromatin; euchromatic repair is unaffected. HP1β depletion rescues the repair defect.","method":"CCAR2 knockout cells, immunofluorescence (γH2AX/53BP1 foci), western blot (Chk2 and KAP1 phosphorylation), epistasis with HP1β depletion","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with specific molecular readout (phosphorylation) and epistasis, single lab","pmids":["26158765"],"is_preprint":false},{"year":2015,"finding":"CCAR2/DBC1 forms a complex with LXRα directly (amino terminus of CCAR2 binds the AF-2 domain of LXRα) in a ligand-independent manner, attenuating LXRα transcriptional activation. CCAR2 competes with SIRT1 for LXRα binding, blocking SIRT1-LXRα complex formation and consequently preventing SIRT1-mediated deacetylation of LXRα.","method":"Co-immunoprecipitation in HepG2 cells, in vitro GST pull-down, competitive immunoprecipitation, RNA interference, target gene expression assay","journal":"The Journal of steroid biochemistry and molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro pull-down plus cellular Co-IP with competitive IP, single lab","pmids":["25661920"],"is_preprint":false},{"year":2015,"finding":"DBC1/CCAR2 promotes stabilization of androgen receptor (AR) protein in osteosarcoma cells by preventing its proteasomal degradation; siRNA knockdown of DBC1 increases poly-ubiquitination and proteasome-mediated degradation of AR.","method":"siRNA knockdown, ubiquitination assay, proteasome inhibitor treatment, western blot, cell invasion and proliferation assays","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mechanistic ubiquitination assay with siRNA and proteasome inhibitor, single lab","pmids":["26249023"],"is_preprint":false},{"year":2016,"finding":"CCAR2 inhibits DNA end resection at double-strand breaks, thereby suppressing homologous recombination (HR) and favouring non-homologous end joining (NHEJ); CCAR2 acts as an antagonist of CtIP-dependent resection. Identified via genome-wide esiRNA screen using the SeeSaw Reporter that distinguishes HR from NHEJ.","method":"Genome-wide esiRNA library screen (SeeSaw Reporter), fluorescence-based HR/NHEJ ratio assay, functional validation of resection by RPA and ssDNA detection","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — genome-wide unbiased screen followed by mechanistic validation of resection inhibition with multiple readouts","pmids":["27503537"],"is_preprint":false},{"year":2016,"finding":"CCAR2 loss inhibits AKT pathway activation specifically in cancer cells (not normal cells) by transcriptionally upregulating TRB3, which binds and inhibits phosphorylation of AKT at Ser473, leading to reduced GSK3β phosphorylation, G1/S arrest, and impaired cancer cell proliferation.","method":"siRNA knockdown, western blot (AKT/pAKT, GSK3β), RT-PCR and protein level measurement of TRB3, co-immunoprecipitation (TRB3–AKT binding), cell cycle analysis","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP showing TRB3–AKT interaction downstream of CCAR2 loss, multiple readouts, single lab","pmids":["27809307"],"is_preprint":false},{"year":2017,"finding":"CCAR2 localizes to mitochondria and forms a complex with the mitochondrial chaperone Hsp60; this interaction increases upon rotenone-induced mitochondrial stress. CCAR2 and Hsp60 co-depletion disrupts mitochondrial membrane potential and promotes apoptosis, indicating the CCAR2–Hsp60 complex supports cell survival during mitochondrial stress.","method":"Affinity purification of CCAR2-containing complexes, co-immunoprecipitation, mitochondrial fractionation, mitochondrial membrane potential assay (JC-1), apoptosis assay, siRNA knockdown","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — subcellular fractionation plus Co-IP demonstrating mitochondrial localization and complex, functional assay, single lab","pmids":["28254432"],"is_preprint":false},{"year":2019,"finding":"N-terminal acetylation of CCAR2 (induced by sulforaphane) diminishes its interactions with HDAC3 and β-catenin, interfering with Wnt co-activator functions. Acetyl-reader proteins BRD9 and BET family members recognise the CCAR2 acetylation sites, establishing a BET/BRD9 acetyl switch. BET inhibitor JQ1 synergizes with sulforaphane in colon cancer cells.","method":"Protein domain arrays, pull-down assays, co-immunoprecipitation, genetically encoded acetylation mimics, tumor prevention model","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pull-down and Co-IP identifying acetyl readers, mutagenesis via genetic acetylation mimics, in vivo tumor model, single lab","pmids":["30643017"],"is_preprint":false},{"year":2019,"finding":"CCAR2 and Hsp60 are both required for survivin expression in neuroblastoma cells; co-depletion of CCAR2 and Hsp60 downregulates survivin (IAP family member), promoting cancer cell death.","method":"siRNA knockdown of CCAR2 and Hsp60, western blot and RT-PCR for survivin, apoptosis assays","journal":"International journal of molecular sciences","confidence":"Low","confidence_rationale":"Tier 3 / Weak — siRNA knockdown with expression readout, no direct biochemical interaction demonstrated between CCAR2 and survivin pathway components, single lab","pmids":["30609639"],"is_preprint":false},{"year":2021,"finding":"Nuclear GAPDH (redistributed by H2S-induced sulfhydration of its active-site cysteine) interacts with CCAR2 in the nucleus, disrupting the inhibitory CCAR2–SIRT1 complex. This activates SIRT1, which deacetylates LC3B, promoting its cytoplasmic translocation and autophagy flux. The pathway restricts intracellular Mycobacterium tuberculosis growth.","method":"Co-immunoprecipitation, proximity ligation assay, nuclear fractionation, siRNA/overexpression, GAPDH active-site cysteine mutant, LC3B deacetylation/localization assay, bacterial CFU assay","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, PLA, mutagenesis of GAPDH active site, multiple orthogonal functional readouts including in vivo bacterial clearance","pmids":["33459133"],"is_preprint":false},{"year":2021,"finding":"CCAR2 is a component of the CECR2-containing chromatin remodeling factor (CERF) complex in embryonic stem (ES) cells but not in the testis, indicating tissue-specific complex assembly. LUZP1 also joins the CERF complex in ES cells and appears to stabilize it.","method":"Mass spectrometry of CECR2 immunoprecipitates from ES cells and testes, co-immunoprecipitation validation","journal":"Biochemistry and cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS-based complex identification confirmed by Co-IP in two tissues, single lab","pmids":["34197713"],"is_preprint":false},{"year":2021,"finding":"CCAR2 acts as a co-activator of Wnt/β-catenin signaling in osteosarcoma cells to drive transcriptional upregulation of SPARC; knockdown of CCAR2 reduces Wnt/β-catenin target gene expression, and forced SPARC expression rescues the malignant phenotype of CCAR2-depleted cells.","method":"siRNA knockdown, transcriptomic profiling of CCAR2-KD cells, rescue experiments (SPARC overexpression), Wnt/β-catenin reporter assay, in vivo xenograft","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — transcriptomic profiling plus functional rescue and in vivo validation, single lab","pmids":["34624572"],"is_preprint":false},{"year":2022,"finding":"CCAR2 is a functional component of the 53BP1–RIF1–Shieldin pathway that restricts DNA double-strand break end-resection and promotes NHEJ. CCAR2 co-immunoprecipitates with the Shieldin complex; its S1-like RNA-binding domain is required for this interaction and for suppression of end-resection. CCAR2 acts downstream of Shieldin (CCAR2 KO delays resolution of Shieldin foci). FHA-domain-dependent targeting of CCAR2 to DSB sites re-sensitizes BRCA1−/−SHLD2−/− cells to PARP inhibitors. CCAR2 KO is epistatic with Shieldin KO.","method":"Co-immunoprecipitation, domain-deletion mutagenesis, CRISPR knockout, RPA/RAD51 loading assays, PARP inhibitor sensitivity assay, epistasis analysis, foci resolution assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — reciprocal Co-IP, domain mutagenesis, genetic epistasis, PARP inhibitor functional rescue, multiple orthogonal approaches in single rigorous study","pmids":["36442094"],"is_preprint":false},{"year":2022,"finding":"CCAR2 governs mitotic progression by spatiotemporally regulating Aurora B kinase activity; CCAR2-deficient cells show premature centromeric cohesion loss, spindle assembly checkpoint inactivation, lagging chromosomes, and activation of the abscission checkpoint, resulting in multilobulated nuclei.","method":"CCAR2 siRNA/knockout, live-cell imaging, immunofluorescence (Aurora B, pH3, BUBR1), chromosome segregation analysis, cytokinesis/abscission checkpoint assay","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with specific mitotic readouts and Aurora B localization data, single lab","pmids":["35672287"],"is_preprint":false},{"year":2025,"finding":"CCAR2 negatively regulates IL-8 production in cervical cancer cells under oxidative stress; CCAR2 depletion activates AP-1 transcription factor, leading to upregulated IL-8 expression.","method":"siRNA knockdown, H2O2 treatment, ELISA and RT-PCR for IL-8, AP-1 reporter/activity assay, patient tissue correlation","journal":"Oncotarget","confidence":"Low","confidence_rationale":"Tier 3 / Weak — siRNA knockdown with cytokine and transcription factor readout, pathway placement via AP-1 reporter, single lab, no direct binding demonstrated","pmids":["29416683"],"is_preprint":false},{"year":2025,"finding":"TFPI2 stabilizes CCAR2 protein by associating with the deubiquitinating enzyme BRCC3, preventing ubiquitination-mediated degradation of CCAR2. CCAR2 in turn stabilizes GADD45A mRNA (via RNA immunoprecipitation), promoting GADD45A-mediated DNA damage and inhibiting homologous recombination repair, thereby sensitizing HCC cells to sorafenib.","method":"RNA immunoprecipitation, chromatin immunoprecipitation, Co-immunoprecipitation (TFPI2–BRCC3–CCAR2), ubiquitination assay, HCC organoids and in vivo models","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple Co-IP/RIP approaches and in vivo validation, single lab, single study","pmids":["40765823"],"is_preprint":false},{"year":2025,"finding":"ASFV p30 viral protein interacts with host CCAR2 (confirmed by co-immunoprecipitation), and both CCAR2 and MATR3 promote ASFV replication; ASFV infection upregulates CCAR2 expression in host cells. CCAR2 and p30 co-localize in the cytoplasm.","method":"Mass spectrometry, co-immunoprecipitation, co-localization (fluorescence microscopy), siRNA knockdown, viral replication assay","journal":"Veterinary microbiology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP confirming interaction, functional siRNA readout, single lab","pmids":["39919500"],"is_preprint":false},{"year":2026,"finding":"UBE4B (E3/E4 ubiquitin ligase) ubiquitinates CCAR2, promoting its proteasomal degradation. UBE4B deficiency leads to CCAR2 accumulation, which inhibits SIRT1 activity, thereby increasing p53 acetylation and stability and promoting apoptosis. UBE4B also directly targets p53 for degradation (dominant pathway), revealed by rescue experiments.","method":"Orthogonal ubiquitin transfer (OUT) screen, co-immunoprecipitation, ubiquitination assay, SIRT1 activity assay, p53 acetylation western blot, transcriptional profiling, rescue experiments","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — OUT substrate screen, in vitro ubiquitination assay, SIRT1 activity measurement, and rescue epistasis, single lab","pmids":["42074320"],"is_preprint":false},{"year":2026,"finding":"CCAR2 binds ERα and alters its nuclear translocation, increasing apoptotic transcriptional activity in osteoclasts, thereby reducing osteoclast numbers. CCAR2 knockout RAW264.7 cells generated by CRISPR-Cas9 show increased osteoclast formation, reduced ROS, and decreased apoptosis during osteoclastogenesis.","method":"CRISPR-Cas9 CCAR2 knockout, osteoclast differentiation assay (RANKL/M-CSF), co-immunoprecipitation (CCAR2–ERα), nuclear translocation assay, apoptosis assay, ROS measurement","journal":"Acta biochimica et biophysica Sinica","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR KO with specific osteoclast phenotype and Co-IP demonstrating CCAR2–ERα interaction, single lab","pmids":["42212624"],"is_preprint":false},{"year":2026,"finding":"Genistein directly binds CCAR2 (binding site identified at Trp108) and disrupts the CCAR2–SIRT1 inhibitory interaction, restoring SIRT1 deacetylase activity; this reduces p53 acetylation and p53-driven pro-fibrotic signaling. Liver-specific Ccar2 deletion recapitulates the hepatoprotective effect, confirming CCAR2 as a direct pharmacological target.","method":"Biotin-conjugated genistein pull-down + mass spectrometry, CETSA, molecular docking, Co-immunoprecipitation, SIRT1 activity assay, p53 acetylation assay, liver-specific Ccar2 KO mouse model","journal":"Phytomedicine","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — pull-down + CETSA identify binding, Co-IP and enzymatic assay confirm mechanism, in vivo KO validation, single lab","pmids":["41775214"],"is_preprint":false},{"year":2026,"finding":"Ginsenoside Rf directly binds CCAR2 and disrupts the CCAR2–SIRT1 interaction, releasing SIRT1 to deacetylate FXR; this promotes FXR nuclear translocation and transcriptional activation of bile acid metabolism genes, protecting against acetaminophen-induced liver injury. Liver-specific Ccar2 deletion attenuates APAP injury and abolishes the ginsenoside Rf hepatoprotective effect.","method":"Pull-down assay (ginsenoside Rf as bait), RNA sequencing, Co-immunoprecipitation (CCAR2–SIRT1), FXR acetylation and nuclear translocation assay, liver-specific Ccar2 KO mice, AILI mouse model","journal":"British journal of pharmacology","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — affinity pull-down, Co-IP, enzymatic substrate (FXR acetylation) assay, in vivo KO validation, single lab","pmids":["41825964"],"is_preprint":false}],"current_model":"CCAR2/DBC1 is a multifunctional nuclear (and, in some contexts, mitochondrial) scaffold protein that acts principally as an endogenous inhibitor of SIRT1 deacetylase by direct binding—an interaction regulated by ATM/ATR-mediated phosphorylation of CCAR2 (promoting binding) and hMOF-mediated acetylation of CCAR2 at K112/K215 (blocking binding)—thereby controlling p53 acetylation, stability, and apoptosis; it also suppresses DNA double-strand break end-resection as a functional component of the 53BP1–RIF1–Shieldin pathway (requiring its S1-like RNA-binding domain), acts as a ligand-independent co-regulator/stabilizer of nuclear receptors including ERα, AR, and LXRα through direct N-terminal domain interactions, participates in Wnt/β-catenin co-activation, regulates mitotic progression via Aurora B, and is itself subject to ubiquitin-mediated proteasomal degradation controlled by UBE4B."},"narrative":{"mechanistic_narrative":"CCAR2 (DBC1) is a multifunctional nuclear scaffold that integrates deacetylase regulation, DNA repair, and nuclear-receptor signaling to control cell survival and genome stability [PMID:24126058, PMID:36442094, PMID:17473282]. Its best-defined activity is as an endogenous inhibitor of the SIRT1 deacetylase: direct CCAR2-SIRT1 binding restrains SIRT1, and this brake is set by post-translational marks and competing binders—hMOF-mediated acetylation of CCAR2 at K112/K215 blocks SIRT1 binding while DNA-damage signaling reduces this acetylation to strengthen the interaction [PMID:24126058], and nuclear GAPDH can displace CCAR2 from SIRT1 to drive SIRT1-dependent LC3B deacetylation and autophagy [PMID:33459133]. By gating SIRT1, CCAR2 controls deacetylation of downstream effectors including p53, with CCAR2 accumulation increasing p53 acetylation, stability, and apoptosis; CCAR2 levels are themselves limited by UBE4B-mediated ubiquitination and proteasomal degradation [PMID:42074320]. In DNA repair, CCAR2 suppresses DNA double-strand break end-resection as a functional component of the 53BP1-RIF1-Shieldin pathway, acting downstream of Shieldin through its S1-like RNA-binding domain to favor NHEJ over homologous recombination [PMID:36442094, PMID:27503537], and it is also required for Chk2-dependent KAP1 phosphorylation needed to repair heterochromatic breaks [PMID:26158765]. CCAR2 additionally functions as a ligand-independent co-regulator and stabilizer of nuclear receptors through N-terminal domain interactions, binding and stabilizing unliganded ERα, stabilizing androgen receptor against degradation, and attenuating LXRα by competing with SIRT1 [PMID:17473282, PMID:26249023, PMID:25661920], and it co-activates Wnt/β-catenin signaling [PMID:34624572]. Beyond the nucleus, CCAR2 localizes to mitochondria and forms a pro-survival complex with Hsp60 under mitochondrial stress [PMID:28254432], and it regulates mitotic progression via spatiotemporal control of Aurora B [PMID:35672287]. The CCAR2-SIRT1 interface is a tractable pharmacological target, disrupted by small molecules including genistein and ginsenoside Rf to restore SIRT1 activity [PMID:41775214, PMID:41825964].","teleology":[{"year":2007,"claim":"Established CCAR2 as a ligand-independent nuclear-receptor co-regulator by showing its N-terminus directly binds and stabilizes unliganded ERα, linking it to hormone-independent breast cancer cell survival.","evidence":"In vitro binding, co-IP, and siRNA with apoptosis readout in ERα-positive breast cancer cells","pmids":["17473282"],"confidence":"High","gaps":["Did not define the structural basis of the N-terminal-ERα interface","Did not connect ERα stabilization to CCAR2's SIRT1-inhibitory role"]},{"year":2013,"claim":"Defined how the CCAR2-SIRT1 brake is regulated, showing hMOF acetylation at K112/K215 blocks SIRT1 binding and DNA damage reverses this to enhance the inhibitory complex.","evidence":"Mass spectrometry, in vitro acetyltransferase assay, mutagenesis, reciprocal co-IP, and apoptosis readout","pmids":["24126058"],"confidence":"High","gaps":["Did not establish in vivo stoichiometry of the acetylation switch","Upstream signals coupling ATM to hMOF inhibition incompletely defined"]},{"year":2015,"claim":"Placed CCAR2 in heterochromatic DSB repair by showing it is required for Chk2-dependent KAP1 phosphorylation and heterochromatin relaxation, separable from euchromatic repair.","evidence":"CCAR2 knockout cells, foci immunofluorescence, phospho-western, and HP1β epistasis","pmids":["26158765"],"confidence":"Medium","gaps":["Mechanism by which CCAR2 promotes Chk2 activation not defined","Single lab without reciprocal genetic confirmation"]},{"year":2015,"claim":"Extended CCAR2's nuclear-receptor co-regulation beyond ERα, showing direct N-terminal binding to LXRα and AR with opposing functional outcomes (LXRα attenuation via SIRT1 competition; AR stabilization against degradation).","evidence":"GST pull-down, competitive co-IP, ubiquitination assay, and siRNA in HepG2 and osteosarcoma cells","pmids":["25661920","26249023"],"confidence":"Medium","gaps":["Why CCAR2 stabilizes some receptors but attenuates others not resolved","Receptor selectivity determinants on the N-terminal domain unknown"]},{"year":2016,"claim":"Identified CCAR2 in an unbiased screen as an inhibitor of DNA end-resection that biases repair toward NHEJ by antagonizing CtIP, establishing a genome-wide functional anchor for its repair role.","evidence":"Genome-wide esiRNA SeeSaw Reporter screen with RPA/ssDNA resection validation","pmids":["27503537"],"confidence":"High","gaps":["Did not yet place CCAR2 in the 53BP1-Shieldin axis","Molecular contact with the resection machinery not defined here"]},{"year":2016,"claim":"Revealed a cancer-selective signaling role in which CCAR2 loss upregulates TRB3 to inhibit AKT Ser473 phosphorylation, causing G1/S arrest.","evidence":"siRNA, western blot of AKT/GSK3β, TRB3-AKT co-IP, and cell cycle analysis","pmids":["27809307"],"confidence":"Medium","gaps":["How CCAR2 transcriptionally controls TRB3 is unclear","Cancer-cell specificity not mechanistically explained"]},{"year":2017,"claim":"Showed CCAR2 has an extranuclear, mitochondrial function, forming a stress-induced complex with Hsp60 that supports membrane potential and survival.","evidence":"Affinity purification, co-IP, mitochondrial fractionation, JC-1, and apoptosis assays under rotenone stress","pmids":["28254432"],"confidence":"Medium","gaps":["Mechanism by which CCAR2-Hsp60 sustains membrane potential undefined","Relationship between mitochondrial and nuclear CCAR2 pools unknown"]},{"year":2019,"claim":"Defined an acetyl-switch governing CCAR2's Wnt co-activator role, where N-terminal acetylation recruits BET/BRD9 readers and weakens HDAC3 and β-catenin binding.","evidence":"Domain arrays, pull-downs, acetylation mimics, and a tumor prevention model with JQ1/sulforaphane synergy","pmids":["30643017"],"confidence":"Medium","gaps":["Enzyme responsible for the relevant N-terminal acetylation in this context not pinned down","Direct structural basis of reader recognition not shown"]},{"year":2019,"claim":"Linked the CCAR2-Hsp60 complex to anti-apoptotic survivin expression in neuroblastoma cells.","evidence":"siRNA co-depletion with survivin western/RT-PCR and apoptosis assays","pmids":["30609639"],"confidence":"Low","gaps":["No direct biochemical interaction between CCAR2 and survivin pathway components shown","Single lab, correlative expression readout only"]},{"year":2021,"claim":"Showed CCAR2 release from SIRT1 can be triggered by nuclear GAPDH redistribution, activating SIRT1-mediated LC3B deacetylation and autophagy to restrict intracellular M. tuberculosis.","evidence":"Reciprocal co-IP, PLA, GAPDH active-site mutant, LC3B assays, and bacterial CFU","pmids":["33459133"],"confidence":"High","gaps":["Physiological stimuli driving GAPDH-CCAR2 engagement beyond H2S not delineated","Direct CCAR2-GAPDH binding interface not mapped"]},{"year":2021,"claim":"Identified CCAR2 as a tissue-specific subunit of the CECR2 (CERF) chromatin remodeling complex in ES cells, broadening its chromatin functions.","evidence":"Mass spectrometry of CECR2 immunoprecipitates from ES cells and testes with co-IP validation","pmids":["34197713"],"confidence":"Medium","gaps":["Functional consequence of CCAR2 within CERF undefined","Determinants of tissue-specific incorporation unknown"]},{"year":2021,"claim":"Established CCAR2 as a Wnt/β-catenin co-activator driving SPARC to promote osteosarcoma malignancy.","evidence":"siRNA, transcriptomics, SPARC rescue, Wnt reporter, and xenograft","pmids":["34624572"],"confidence":"Medium","gaps":["Direct promoter-level mechanism of SPARC induction not shown","Relation to the BET/BRD9 acetyl switch not tested"]},{"year":2022,"claim":"Resolved CCAR2's resection-suppressing activity into the 53BP1-RIF1-Shieldin pathway, acting downstream of Shieldin via its S1-like RNA-binding domain to promote NHEJ.","evidence":"Reciprocal co-IP, domain-deletion mutagenesis, CRISPR KO, RPA/RAD51 assays, epistasis, and PARP-inhibitor rescue in BRCA1−/−SHLD2−/− cells","pmids":["36442094"],"confidence":"High","gaps":["RNA ligand (if any) bound by the S1-like domain not identified","Structural basis of Shieldin engagement undefined"]},{"year":2022,"claim":"Demonstrated a mitotic role for CCAR2 in spatiotemporal control of Aurora B, required for proper centromeric cohesion, checkpoint signaling, and faithful segregation.","evidence":"siRNA/KO, live-cell imaging, Aurora B/BUBR1 immunofluorescence, and abscission checkpoint assays","pmids":["35672287"],"confidence":"Medium","gaps":["Whether CCAR2 directly binds or regulates Aurora B not established","Connection to its nuclear scaffold functions unclear"]},{"year":2025,"claim":"Connected CCAR2 to oxidative-stress cytokine control, with its depletion activating AP-1 and IL-8 in cervical cancer cells.","evidence":"siRNA, H2O2 treatment, IL-8 ELISA/RT-PCR, AP-1 reporter, and patient tissue correlation","pmids":["29416683"],"confidence":"Low","gaps":["No direct binding linking CCAR2 to the AP-1/IL-8 axis demonstrated","Single lab, pathway placement inferred from reporter only"]},{"year":2025,"claim":"Showed CCAR2 protein stability is controlled by a TFPI2-BRCC3 deubiquitination axis and that CCAR2 stabilizes GADD45A mRNA to inhibit HR and sensitize HCC to sorafenib.","evidence":"RNA-IP, ChIP, co-IP (TFPI2-BRCC3-CCAR2), ubiquitination assay, organoids and in vivo models","pmids":["40765823"],"confidence":"Medium","gaps":["Direct CCAR2-GADD45A mRNA binding determinants not mapped","Interplay with the UBE4B degradation pathway untested"]},{"year":2025,"claim":"Reported a cytoplasmic CCAR2-p30 interaction in which CCAR2 supports African swine fever virus replication.","evidence":"Mass spectrometry, co-IP, co-localization, siRNA, and viral replication assay","pmids":["39919500"],"confidence":"Low","gaps":["Single co-IP without reciprocal/structural validation","Mechanism by which CCAR2 aids replication unknown"]},{"year":2026,"claim":"Identified UBE4B as an E3/E4 ligase that ubiquitinates CCAR2 for degradation, linking CCAR2 turnover to SIRT1 activity and p53 acetylation/apoptosis.","evidence":"OUT substrate screen, in vitro ubiquitination, SIRT1 activity assay, p53 acetylation western, and rescue epistasis","pmids":["42074320"],"confidence":"Medium","gaps":["Relative contribution of CCAR2 versus direct p53 degradation by UBE4B context-dependent","Signals regulating UBE4B-CCAR2 targeting unknown"]},{"year":2026,"claim":"Validated the CCAR2-SIRT1 interface as a druggable target by showing distinct natural products disrupt the inhibitory complex to restore SIRT1-dependent deacetylation of p53, FXR, and pro-survival programs in liver disease.","evidence":"Affinity pull-down/MS, CETSA, docking, co-IP, substrate-acetylation assays, and liver-specific Ccar2 KO mouse models","pmids":["41775214","41825964"],"confidence":"Medium","gaps":["Co-crystal structures of compound-bound CCAR2 not reported","Selectivity over other CCAR2 functions not assessed"]},{"year":null,"claim":"How CCAR2's diverse activities—SIRT1 inhibition, Shieldin-dependent resection control, nuclear-receptor stabilization, chromatin remodeling, mitotic regulation, and mitochondrial survival signaling—are coordinated within a single protein, and which are governed by shared post-translational switches, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No integrated structural model linking CCAR2's domains to its multiple complexes","Switching between nuclear, mitochondrial, and repair pools not mechanistically explained","Whether the S1-like RNA-binding domain binds a defined RNA is unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[1,18,20,21]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,3,12]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[13,16]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,4,13]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1,10,13]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[7]},{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[2,13]}],"pathway":[{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[2,5,13]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,3,12]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[1,18]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[10]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[14]}],"complexes":["53BP1-RIF1-Shieldin complex","CECR2-containing remodeling factor (CERF) complex","CCAR2-SIRT1 complex","CCAR2-Hsp60 complex"],"partners":["SIRT1","ESR1","AR","NR1H3","HSPD1","GAPDH","CTNNB1","UBE4B"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8N163","full_name":"Cell cycle and apoptosis regulator protein 2","aliases":["Cell division cycle and apoptosis regulator protein 2","DBIRD complex subunit KIAA1967","Deleted in breast cancer gene 1 protein","DBC-1","DBC.1","NET35","p30 DBC"],"length_aa":923,"mass_kda":102.9,"function":"Core component of the DBIRD complex, a multiprotein complex that acts at the interface between core mRNP particles and RNA polymerase II (RNAPII) and integrates transcript elongation with the regulation of alternative splicing: the DBIRD complex affects local transcript elongation rates and alternative splicing of a large set of exons embedded in (A + T)-rich DNA regions (PubMed:22446626). Inhibits SIRT1 deacetylase activity leading to increasing levels of p53/TP53 acetylation and p53-mediated apoptosis (PubMed:18235501, PubMed:18235502, PubMed:23352644). Inhibits SUV39H1 methyltransferase activity (PubMed:19218236). Mediates ligand-dependent transcriptional activation by nuclear hormone receptors (PubMed:19131338). Plays a critical role in maintaining genomic stability and cellular integrity following UV-induced genotoxic stress (PubMed:23398316). Regulates the circadian expression of the core clock components NR1D1 and BMAL1 (PubMed:23398316). Enhances the transcriptional repressor activity of NR1D1 through stabilization of NR1D1 protein levels by preventing its ubiquitination and subsequent degradation (PubMed:23398316). Represses the ligand-dependent transcriptional activation function of ESR2 (PubMed:20074560). Acts as a regulator of PCK1 expression and gluconeogenesis by a mechanism that involves, at least in part, both NR1D1 and SIRT1 (PubMed:24415752). Negatively regulates the deacetylase activity of HDAC3 and can alter its subcellular localization (PubMed:21030595). Positively regulates the beta-catenin pathway (canonical Wnt signaling pathway) and is required for MCC-mediated repression of the beta-catenin pathway (PubMed:24824780). Represses ligand-dependent transcriptional activation function of NR1H2 and NR1H3 and inhibits the interaction of SIRT1 with NR1H3 (PubMed:25661920). Plays an important role in tumor suppression through p53/TP53 regulation; stabilizes p53/TP53 by affecting its interaction with ubiquitin ligase MDM2 (PubMed:25732823). Represses the transcriptional activator activity of BRCA1 (PubMed:20160719). Inhibits SIRT1 in a CHEK2 and PSEM3-dependent manner and inhibits the activity of CHEK2 in vitro (PubMed:25361978)","subcellular_location":"Nucleus; Cytoplasm; Cytoplasm, cytoskeleton, spindle","url":"https://www.uniprot.org/uniprotkb/Q8N163/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CCAR2","classification":"Not Classified","n_dependent_lines":6,"n_total_lines":1208,"dependency_fraction":0.004966887417218543},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"DDX21","stoichiometry":0.2},{"gene":"HNRNPL","stoichiometry":0.2},{"gene":"UBA52","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/CCAR2","total_profiled":1310},"omim":[{"mim_id":"607359","title":"CELL DIVISION CYCLE AND APOPTOSIS REGULATOR 2; CCAR2","url":"https://www.omim.org/entry/607359"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nucleoplasm","reliability":"Enhanced"},{"location":"Nuclear bodies","reliability":"Enhanced"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CCAR2"},"hgnc":{"alias_symbol":["DBC-1","DBC1","NET35"],"prev_symbol":["KIAA1967"]},"alphafold":{"accession":"Q8N163","domains":[{"cath_id":"2.40.50.140","chopping":"56-116","consensus_level":"high","plddt":89.0728,"start":56,"end":116},{"cath_id":"3.90.79.10","chopping":"238-288_300-329_338-445_506-553","consensus_level":"high","plddt":90.3685,"start":238,"end":553},{"cath_id":"1.20.5","chopping":"812-916","consensus_level":"medium","plddt":90.4872,"start":812,"end":916}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8N163","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8N163-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8N163-F1-predicted_aligned_error_v6.png","plddt_mean":68.81},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CCAR2","jax_strain_url":"https://www.jax.org/strain/search?query=CCAR2"},"sequence":{"accession":"Q8N163","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8N163.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8N163/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8N163"}},"corpus_meta":[{"pmid":"17473282","id":"PMC_17473282","title":"Modulation 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communications","url":"https://pubmed.ncbi.nlm.nih.gov/28254432","citation_count":11,"is_preprint":false},{"pmid":"34197713","id":"PMC_34197713","title":"Chromatin remodeling factor CECR2 forms tissue-specific complexes with CCAR2 and LUZP1.","date":"2021","source":"Biochemistry and cell biology = Biochimie et biologie cellulaire","url":"https://pubmed.ncbi.nlm.nih.gov/34197713","citation_count":8,"is_preprint":false},{"pmid":"36434857","id":"PMC_36434857","title":"MiR-342-5p protects neurons from cerebral ischemia induced-apoptosis through regulation of Akt/NF-κB pathways by targeting CCAR2.","date":"2022","source":"Journal of stroke and cerebrovascular diseases : the official journal of National Stroke Association","url":"https://pubmed.ncbi.nlm.nih.gov/36434857","citation_count":8,"is_preprint":false},{"pmid":"34624572","id":"PMC_34624572","title":"CCAR2 promotes a malignant phenotype of osteosarcoma through Wnt/β-catenin-dependent transcriptional activation of 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targets CCAR2 to induce neuronal oxidative stress and apoptosis via mitochondria-mediated pathways following traumatic brain injury.","date":"2024","source":"Free radical biology & medicine","url":"https://pubmed.ncbi.nlm.nih.gov/39709098","citation_count":5,"is_preprint":false},{"pmid":"39612069","id":"PMC_39612069","title":"CircKRT75 augments the cisplatin chemoresistance of nasopharyngeal carcinoma via targeting miR-659/CCAR2 axis.","date":"2024","source":"Journal of molecular histology","url":"https://pubmed.ncbi.nlm.nih.gov/39612069","citation_count":4,"is_preprint":false},{"pmid":"40765823","id":"PMC_40765823","title":"Tissue Factor Pathway Inhibitor 2 Enhances Hepatocellular Carcinoma Chemosensitivity by Activating CCAR2-GADD45A-Mediated DNA Damage Repair.","date":"2025","source":"International journal of biological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/40765823","citation_count":3,"is_preprint":false},{"pmid":"40980968","id":"PMC_40980968","title":"Kaempferol 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pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/41825964","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":20080,"output_tokens":6004,"usd":0.07515,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":14848,"output_tokens":6224,"usd":0.11492,"stage2_stop_reason":"end_turn"},"total_usd":0.19007,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2007,\n      \"finding\": \"DBC-1/CCAR2 amino terminus binds directly to the ERα hormone-binding domain in a ligand-independent manner, stabilizing unliganded ERα protein; antiestrogens (tamoxifen, ICI 182,780) and estrogen disrupt this interaction. RNAi-mediated DBC-1 depletion reduces unliganded ERα protein levels and promotes hormone-independent apoptosis of ERα-positive breast cancer cells.\",\n      \"method\": \"In vitro binding assay, co-immunoprecipitation, RNA interference (siRNA knockdown), apoptosis assays\",\n      \"journal\": \"Molecular endocrinology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — direct in vitro binding plus in vivo co-IP, RNAi functional readout with specific phenotype, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"17473282\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"hMOF (MYST-family acetyltransferase) acetylates DBC1/CCAR2 on lysine residues K112 and K215; this acetylation inhibits DBC1 binding to SIRT1, thereby increasing SIRT1 deacetylase activity. SIRT1 in turn promotes DBC1 deacetylation, forming a negative-feedback loop. After DNA damage, ATM-dependent inhibition of hMOF binding to DBC1 reduces DBC1 acetylation and enhances DBC1–SIRT1 binding. A DBC1 acetylation-mimic mutant fails to promote apoptosis after DNA damage.\",\n      \"method\": \"Mass spectrometry, co-immunoprecipitation, in vitro acetyltransferase assay, site-directed mutagenesis, deacetylase activity assay, apoptosis assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro enzymatic assay, MS identification of sites, mutagenesis, reciprocal Co-IP, functional apoptosis readout in single rigorous study\",\n      \"pmids\": [\"24126058\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CCAR2 is required for Chk2-dependent phosphorylation of KAP1, a step needed for heterochromatin relaxation and repair of heterochromatic DNA double-strand breaks. CCAR2 knockout cells exhibit defective Chk2 activation, elevated KAP1 non-phosphorylation, and persistent DNA damage foci specifically in heterochromatin; euchromatic repair is unaffected. HP1β depletion rescues the repair defect.\",\n      \"method\": \"CCAR2 knockout cells, immunofluorescence (γH2AX/53BP1 foci), western blot (Chk2 and KAP1 phosphorylation), epistasis with HP1β depletion\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with specific molecular readout (phosphorylation) and epistasis, single lab\",\n      \"pmids\": [\"26158765\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CCAR2/DBC1 forms a complex with LXRα directly (amino terminus of CCAR2 binds the AF-2 domain of LXRα) in a ligand-independent manner, attenuating LXRα transcriptional activation. CCAR2 competes with SIRT1 for LXRα binding, blocking SIRT1-LXRα complex formation and consequently preventing SIRT1-mediated deacetylation of LXRα.\",\n      \"method\": \"Co-immunoprecipitation in HepG2 cells, in vitro GST pull-down, competitive immunoprecipitation, RNA interference, target gene expression assay\",\n      \"journal\": \"The Journal of steroid biochemistry and molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro pull-down plus cellular Co-IP with competitive IP, single lab\",\n      \"pmids\": [\"25661920\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"DBC1/CCAR2 promotes stabilization of androgen receptor (AR) protein in osteosarcoma cells by preventing its proteasomal degradation; siRNA knockdown of DBC1 increases poly-ubiquitination and proteasome-mediated degradation of AR.\",\n      \"method\": \"siRNA knockdown, ubiquitination assay, proteasome inhibitor treatment, western blot, cell invasion and proliferation assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mechanistic ubiquitination assay with siRNA and proteasome inhibitor, single lab\",\n      \"pmids\": [\"26249023\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CCAR2 inhibits DNA end resection at double-strand breaks, thereby suppressing homologous recombination (HR) and favouring non-homologous end joining (NHEJ); CCAR2 acts as an antagonist of CtIP-dependent resection. Identified via genome-wide esiRNA screen using the SeeSaw Reporter that distinguishes HR from NHEJ.\",\n      \"method\": \"Genome-wide esiRNA library screen (SeeSaw Reporter), fluorescence-based HR/NHEJ ratio assay, functional validation of resection by RPA and ssDNA detection\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genome-wide unbiased screen followed by mechanistic validation of resection inhibition with multiple readouts\",\n      \"pmids\": [\"27503537\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CCAR2 loss inhibits AKT pathway activation specifically in cancer cells (not normal cells) by transcriptionally upregulating TRB3, which binds and inhibits phosphorylation of AKT at Ser473, leading to reduced GSK3β phosphorylation, G1/S arrest, and impaired cancer cell proliferation.\",\n      \"method\": \"siRNA knockdown, western blot (AKT/pAKT, GSK3β), RT-PCR and protein level measurement of TRB3, co-immunoprecipitation (TRB3–AKT binding), cell cycle analysis\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP showing TRB3–AKT interaction downstream of CCAR2 loss, multiple readouts, single lab\",\n      \"pmids\": [\"27809307\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CCAR2 localizes to mitochondria and forms a complex with the mitochondrial chaperone Hsp60; this interaction increases upon rotenone-induced mitochondrial stress. CCAR2 and Hsp60 co-depletion disrupts mitochondrial membrane potential and promotes apoptosis, indicating the CCAR2–Hsp60 complex supports cell survival during mitochondrial stress.\",\n      \"method\": \"Affinity purification of CCAR2-containing complexes, co-immunoprecipitation, mitochondrial fractionation, mitochondrial membrane potential assay (JC-1), apoptosis assay, siRNA knockdown\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — subcellular fractionation plus Co-IP demonstrating mitochondrial localization and complex, functional assay, single lab\",\n      \"pmids\": [\"28254432\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"N-terminal acetylation of CCAR2 (induced by sulforaphane) diminishes its interactions with HDAC3 and β-catenin, interfering with Wnt co-activator functions. Acetyl-reader proteins BRD9 and BET family members recognise the CCAR2 acetylation sites, establishing a BET/BRD9 acetyl switch. BET inhibitor JQ1 synergizes with sulforaphane in colon cancer cells.\",\n      \"method\": \"Protein domain arrays, pull-down assays, co-immunoprecipitation, genetically encoded acetylation mimics, tumor prevention model\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pull-down and Co-IP identifying acetyl readers, mutagenesis via genetic acetylation mimics, in vivo tumor model, single lab\",\n      \"pmids\": [\"30643017\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CCAR2 and Hsp60 are both required for survivin expression in neuroblastoma cells; co-depletion of CCAR2 and Hsp60 downregulates survivin (IAP family member), promoting cancer cell death.\",\n      \"method\": \"siRNA knockdown of CCAR2 and Hsp60, western blot and RT-PCR for survivin, apoptosis assays\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — siRNA knockdown with expression readout, no direct biochemical interaction demonstrated between CCAR2 and survivin pathway components, single lab\",\n      \"pmids\": [\"30609639\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Nuclear GAPDH (redistributed by H2S-induced sulfhydration of its active-site cysteine) interacts with CCAR2 in the nucleus, disrupting the inhibitory CCAR2–SIRT1 complex. This activates SIRT1, which deacetylates LC3B, promoting its cytoplasmic translocation and autophagy flux. The pathway restricts intracellular Mycobacterium tuberculosis growth.\",\n      \"method\": \"Co-immunoprecipitation, proximity ligation assay, nuclear fractionation, siRNA/overexpression, GAPDH active-site cysteine mutant, LC3B deacetylation/localization assay, bacterial CFU assay\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, PLA, mutagenesis of GAPDH active site, multiple orthogonal functional readouts including in vivo bacterial clearance\",\n      \"pmids\": [\"33459133\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CCAR2 is a component of the CECR2-containing chromatin remodeling factor (CERF) complex in embryonic stem (ES) cells but not in the testis, indicating tissue-specific complex assembly. LUZP1 also joins the CERF complex in ES cells and appears to stabilize it.\",\n      \"method\": \"Mass spectrometry of CECR2 immunoprecipitates from ES cells and testes, co-immunoprecipitation validation\",\n      \"journal\": \"Biochemistry and cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-based complex identification confirmed by Co-IP in two tissues, single lab\",\n      \"pmids\": [\"34197713\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CCAR2 acts as a co-activator of Wnt/β-catenin signaling in osteosarcoma cells to drive transcriptional upregulation of SPARC; knockdown of CCAR2 reduces Wnt/β-catenin target gene expression, and forced SPARC expression rescues the malignant phenotype of CCAR2-depleted cells.\",\n      \"method\": \"siRNA knockdown, transcriptomic profiling of CCAR2-KD cells, rescue experiments (SPARC overexpression), Wnt/β-catenin reporter assay, in vivo xenograft\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — transcriptomic profiling plus functional rescue and in vivo validation, single lab\",\n      \"pmids\": [\"34624572\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CCAR2 is a functional component of the 53BP1–RIF1–Shieldin pathway that restricts DNA double-strand break end-resection and promotes NHEJ. CCAR2 co-immunoprecipitates with the Shieldin complex; its S1-like RNA-binding domain is required for this interaction and for suppression of end-resection. CCAR2 acts downstream of Shieldin (CCAR2 KO delays resolution of Shieldin foci). FHA-domain-dependent targeting of CCAR2 to DSB sites re-sensitizes BRCA1−/−SHLD2−/− cells to PARP inhibitors. CCAR2 KO is epistatic with Shieldin KO.\",\n      \"method\": \"Co-immunoprecipitation, domain-deletion mutagenesis, CRISPR knockout, RPA/RAD51 loading assays, PARP inhibitor sensitivity assay, epistasis analysis, foci resolution assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — reciprocal Co-IP, domain mutagenesis, genetic epistasis, PARP inhibitor functional rescue, multiple orthogonal approaches in single rigorous study\",\n      \"pmids\": [\"36442094\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CCAR2 governs mitotic progression by spatiotemporally regulating Aurora B kinase activity; CCAR2-deficient cells show premature centromeric cohesion loss, spindle assembly checkpoint inactivation, lagging chromosomes, and activation of the abscission checkpoint, resulting in multilobulated nuclei.\",\n      \"method\": \"CCAR2 siRNA/knockout, live-cell imaging, immunofluorescence (Aurora B, pH3, BUBR1), chromosome segregation analysis, cytokinesis/abscission checkpoint assay\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with specific mitotic readouts and Aurora B localization data, single lab\",\n      \"pmids\": [\"35672287\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CCAR2 negatively regulates IL-8 production in cervical cancer cells under oxidative stress; CCAR2 depletion activates AP-1 transcription factor, leading to upregulated IL-8 expression.\",\n      \"method\": \"siRNA knockdown, H2O2 treatment, ELISA and RT-PCR for IL-8, AP-1 reporter/activity assay, patient tissue correlation\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — siRNA knockdown with cytokine and transcription factor readout, pathway placement via AP-1 reporter, single lab, no direct binding demonstrated\",\n      \"pmids\": [\"29416683\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TFPI2 stabilizes CCAR2 protein by associating with the deubiquitinating enzyme BRCC3, preventing ubiquitination-mediated degradation of CCAR2. CCAR2 in turn stabilizes GADD45A mRNA (via RNA immunoprecipitation), promoting GADD45A-mediated DNA damage and inhibiting homologous recombination repair, thereby sensitizing HCC cells to sorafenib.\",\n      \"method\": \"RNA immunoprecipitation, chromatin immunoprecipitation, Co-immunoprecipitation (TFPI2–BRCC3–CCAR2), ubiquitination assay, HCC organoids and in vivo models\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple Co-IP/RIP approaches and in vivo validation, single lab, single study\",\n      \"pmids\": [\"40765823\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ASFV p30 viral protein interacts with host CCAR2 (confirmed by co-immunoprecipitation), and both CCAR2 and MATR3 promote ASFV replication; ASFV infection upregulates CCAR2 expression in host cells. CCAR2 and p30 co-localize in the cytoplasm.\",\n      \"method\": \"Mass spectrometry, co-immunoprecipitation, co-localization (fluorescence microscopy), siRNA knockdown, viral replication assay\",\n      \"journal\": \"Veterinary microbiology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP confirming interaction, functional siRNA readout, single lab\",\n      \"pmids\": [\"39919500\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"UBE4B (E3/E4 ubiquitin ligase) ubiquitinates CCAR2, promoting its proteasomal degradation. UBE4B deficiency leads to CCAR2 accumulation, which inhibits SIRT1 activity, thereby increasing p53 acetylation and stability and promoting apoptosis. UBE4B also directly targets p53 for degradation (dominant pathway), revealed by rescue experiments.\",\n      \"method\": \"Orthogonal ubiquitin transfer (OUT) screen, co-immunoprecipitation, ubiquitination assay, SIRT1 activity assay, p53 acetylation western blot, transcriptional profiling, rescue experiments\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — OUT substrate screen, in vitro ubiquitination assay, SIRT1 activity measurement, and rescue epistasis, single lab\",\n      \"pmids\": [\"42074320\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"CCAR2 binds ERα and alters its nuclear translocation, increasing apoptotic transcriptional activity in osteoclasts, thereby reducing osteoclast numbers. CCAR2 knockout RAW264.7 cells generated by CRISPR-Cas9 show increased osteoclast formation, reduced ROS, and decreased apoptosis during osteoclastogenesis.\",\n      \"method\": \"CRISPR-Cas9 CCAR2 knockout, osteoclast differentiation assay (RANKL/M-CSF), co-immunoprecipitation (CCAR2–ERα), nuclear translocation assay, apoptosis assay, ROS measurement\",\n      \"journal\": \"Acta biochimica et biophysica Sinica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR KO with specific osteoclast phenotype and Co-IP demonstrating CCAR2–ERα interaction, single lab\",\n      \"pmids\": [\"42212624\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Genistein directly binds CCAR2 (binding site identified at Trp108) and disrupts the CCAR2–SIRT1 inhibitory interaction, restoring SIRT1 deacetylase activity; this reduces p53 acetylation and p53-driven pro-fibrotic signaling. Liver-specific Ccar2 deletion recapitulates the hepatoprotective effect, confirming CCAR2 as a direct pharmacological target.\",\n      \"method\": \"Biotin-conjugated genistein pull-down + mass spectrometry, CETSA, molecular docking, Co-immunoprecipitation, SIRT1 activity assay, p53 acetylation assay, liver-specific Ccar2 KO mouse model\",\n      \"journal\": \"Phytomedicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — pull-down + CETSA identify binding, Co-IP and enzymatic assay confirm mechanism, in vivo KO validation, single lab\",\n      \"pmids\": [\"41775214\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Ginsenoside Rf directly binds CCAR2 and disrupts the CCAR2–SIRT1 interaction, releasing SIRT1 to deacetylate FXR; this promotes FXR nuclear translocation and transcriptional activation of bile acid metabolism genes, protecting against acetaminophen-induced liver injury. Liver-specific Ccar2 deletion attenuates APAP injury and abolishes the ginsenoside Rf hepatoprotective effect.\",\n      \"method\": \"Pull-down assay (ginsenoside Rf as bait), RNA sequencing, Co-immunoprecipitation (CCAR2–SIRT1), FXR acetylation and nuclear translocation assay, liver-specific Ccar2 KO mice, AILI mouse model\",\n      \"journal\": \"British journal of pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — affinity pull-down, Co-IP, enzymatic substrate (FXR acetylation) assay, in vivo KO validation, single lab\",\n      \"pmids\": [\"41825964\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CCAR2/DBC1 is a multifunctional nuclear (and, in some contexts, mitochondrial) scaffold protein that acts principally as an endogenous inhibitor of SIRT1 deacetylase by direct binding—an interaction regulated by ATM/ATR-mediated phosphorylation of CCAR2 (promoting binding) and hMOF-mediated acetylation of CCAR2 at K112/K215 (blocking binding)—thereby controlling p53 acetylation, stability, and apoptosis; it also suppresses DNA double-strand break end-resection as a functional component of the 53BP1–RIF1–Shieldin pathway (requiring its S1-like RNA-binding domain), acts as a ligand-independent co-regulator/stabilizer of nuclear receptors including ERα, AR, and LXRα through direct N-terminal domain interactions, participates in Wnt/β-catenin co-activation, regulates mitotic progression via Aurora B, and is itself subject to ubiquitin-mediated proteasomal degradation controlled by UBE4B.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CCAR2 (DBC1) is a multifunctional nuclear scaffold that integrates deacetylase regulation, DNA repair, and nuclear-receptor signaling to control cell survival and genome stability [#1, #13, #0]. Its best-defined activity is as an endogenous inhibitor of the SIRT1 deacetylase: direct CCAR2-SIRT1 binding restrains SIRT1, and this brake is set by post-translational marks and competing binders\\u2014hMOF-mediated acetylation of CCAR2 at K112/K215 blocks SIRT1 binding while DNA-damage signaling reduces this acetylation to strengthen the interaction [#1], and nuclear GAPDH can displace CCAR2 from SIRT1 to drive SIRT1-dependent LC3B deacetylation and autophagy [#10]. By gating SIRT1, CCAR2 controls deacetylation of downstream effectors including p53, with CCAR2 accumulation increasing p53 acetylation, stability, and apoptosis; CCAR2 levels are themselves limited by UBE4B-mediated ubiquitination and proteasomal degradation [#18]. In DNA repair, CCAR2 suppresses DNA double-strand break end-resection as a functional component of the 53BP1-RIF1-Shieldin pathway, acting downstream of Shieldin through its S1-like RNA-binding domain to favor NHEJ over homologous recombination [#13, #5], and it is also required for Chk2-dependent KAP1 phosphorylation needed to repair heterochromatic breaks [#2]. CCAR2 additionally functions as a ligand-independent co-regulator and stabilizer of nuclear receptors through N-terminal domain interactions, binding and stabilizing unliganded ER\\u03b1, stabilizing androgen receptor against degradation, and attenuating LXR\\u03b1 by competing with SIRT1 [#0, #4, #3], and it co-activates Wnt/\\u03b2-catenin signaling [#12]. Beyond the nucleus, CCAR2 localizes to mitochondria and forms a pro-survival complex with Hsp60 under mitochondrial stress [#7], and it regulates mitotic progression via spatiotemporal control of Aurora B [#14]. The CCAR2-SIRT1 interface is a tractable pharmacological target, disrupted by small molecules including genistein and ginsenoside Rf to restore SIRT1 activity [#20, #21].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Established CCAR2 as a ligand-independent nuclear-receptor co-regulator by showing its N-terminus directly binds and stabilizes unliganded ER\\u03b1, linking it to hormone-independent breast cancer cell survival.\",\n      \"evidence\": \"In vitro binding, co-IP, and siRNA with apoptosis readout in ER\\u03b1-positive breast cancer cells\",\n      \"pmids\": [\"17473282\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the structural basis of the N-terminal-ER\\u03b1 interface\", \"Did not connect ER\\u03b1 stabilization to CCAR2's SIRT1-inhibitory role\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defined how the CCAR2-SIRT1 brake is regulated, showing hMOF acetylation at K112/K215 blocks SIRT1 binding and DNA damage reverses this to enhance the inhibitory complex.\",\n      \"evidence\": \"Mass spectrometry, in vitro acetyltransferase assay, mutagenesis, reciprocal co-IP, and apoptosis readout\",\n      \"pmids\": [\"24126058\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish in vivo stoichiometry of the acetylation switch\", \"Upstream signals coupling ATM to hMOF inhibition incompletely defined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Placed CCAR2 in heterochromatic DSB repair by showing it is required for Chk2-dependent KAP1 phosphorylation and heterochromatin relaxation, separable from euchromatic repair.\",\n      \"evidence\": \"CCAR2 knockout cells, foci immunofluorescence, phospho-western, and HP1\\u03b2 epistasis\",\n      \"pmids\": [\"26158765\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which CCAR2 promotes Chk2 activation not defined\", \"Single lab without reciprocal genetic confirmation\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Extended CCAR2's nuclear-receptor co-regulation beyond ER\\u03b1, showing direct N-terminal binding to LXR\\u03b1 and AR with opposing functional outcomes (LXR\\u03b1 attenuation via SIRT1 competition; AR stabilization against degradation).\",\n      \"evidence\": \"GST pull-down, competitive co-IP, ubiquitination assay, and siRNA in HepG2 and osteosarcoma cells\",\n      \"pmids\": [\"25661920\", \"26249023\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Why CCAR2 stabilizes some receptors but attenuates others not resolved\", \"Receptor selectivity determinants on the N-terminal domain unknown\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identified CCAR2 in an unbiased screen as an inhibitor of DNA end-resection that biases repair toward NHEJ by antagonizing CtIP, establishing a genome-wide functional anchor for its repair role.\",\n      \"evidence\": \"Genome-wide esiRNA SeeSaw Reporter screen with RPA/ssDNA resection validation\",\n      \"pmids\": [\"27503537\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not yet place CCAR2 in the 53BP1-Shieldin axis\", \"Molecular contact with the resection machinery not defined here\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Revealed a cancer-selective signaling role in which CCAR2 loss upregulates TRB3 to inhibit AKT Ser473 phosphorylation, causing G1/S arrest.\",\n      \"evidence\": \"siRNA, western blot of AKT/GSK3\\u03b2, TRB3-AKT co-IP, and cell cycle analysis\",\n      \"pmids\": [\"27809307\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How CCAR2 transcriptionally controls TRB3 is unclear\", \"Cancer-cell specificity not mechanistically explained\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showed CCAR2 has an extranuclear, mitochondrial function, forming a stress-induced complex with Hsp60 that supports membrane potential and survival.\",\n      \"evidence\": \"Affinity purification, co-IP, mitochondrial fractionation, JC-1, and apoptosis assays under rotenone stress\",\n      \"pmids\": [\"28254432\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which CCAR2-Hsp60 sustains membrane potential undefined\", \"Relationship between mitochondrial and nuclear CCAR2 pools unknown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined an acetyl-switch governing CCAR2's Wnt co-activator role, where N-terminal acetylation recruits BET/BRD9 readers and weakens HDAC3 and \\u03b2-catenin binding.\",\n      \"evidence\": \"Domain arrays, pull-downs, acetylation mimics, and a tumor prevention model with JQ1/sulforaphane synergy\",\n      \"pmids\": [\"30643017\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Enzyme responsible for the relevant N-terminal acetylation in this context not pinned down\", \"Direct structural basis of reader recognition not shown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Linked the CCAR2-Hsp60 complex to anti-apoptotic survivin expression in neuroblastoma cells.\",\n      \"evidence\": \"siRNA co-depletion with survivin western/RT-PCR and apoptosis assays\",\n      \"pmids\": [\"30609639\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No direct biochemical interaction between CCAR2 and survivin pathway components shown\", \"Single lab, correlative expression readout only\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Showed CCAR2 release from SIRT1 can be triggered by nuclear GAPDH redistribution, activating SIRT1-mediated LC3B deacetylation and autophagy to restrict intracellular M. tuberculosis.\",\n      \"evidence\": \"Reciprocal co-IP, PLA, GAPDH active-site mutant, LC3B assays, and bacterial CFU\",\n      \"pmids\": [\"33459133\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological stimuli driving GAPDH-CCAR2 engagement beyond H2S not delineated\", \"Direct CCAR2-GAPDH binding interface not mapped\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identified CCAR2 as a tissue-specific subunit of the CECR2 (CERF) chromatin remodeling complex in ES cells, broadening its chromatin functions.\",\n      \"evidence\": \"Mass spectrometry of CECR2 immunoprecipitates from ES cells and testes with co-IP validation\",\n      \"pmids\": [\"34197713\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of CCAR2 within CERF undefined\", \"Determinants of tissue-specific incorporation unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Established CCAR2 as a Wnt/\\u03b2-catenin co-activator driving SPARC to promote osteosarcoma malignancy.\",\n      \"evidence\": \"siRNA, transcriptomics, SPARC rescue, Wnt reporter, and xenograft\",\n      \"pmids\": [\"34624572\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct promoter-level mechanism of SPARC induction not shown\", \"Relation to the BET/BRD9 acetyl switch not tested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Resolved CCAR2's resection-suppressing activity into the 53BP1-RIF1-Shieldin pathway, acting downstream of Shieldin via its S1-like RNA-binding domain to promote NHEJ.\",\n      \"evidence\": \"Reciprocal co-IP, domain-deletion mutagenesis, CRISPR KO, RPA/RAD51 assays, epistasis, and PARP-inhibitor rescue in BRCA1\\u2212/\\u2212SHLD2\\u2212/\\u2212 cells\",\n      \"pmids\": [\"36442094\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"RNA ligand (if any) bound by the S1-like domain not identified\", \"Structural basis of Shieldin engagement undefined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Demonstrated a mitotic role for CCAR2 in spatiotemporal control of Aurora B, required for proper centromeric cohesion, checkpoint signaling, and faithful segregation.\",\n      \"evidence\": \"siRNA/KO, live-cell imaging, Aurora B/BUBR1 immunofluorescence, and abscission checkpoint assays\",\n      \"pmids\": [\"35672287\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether CCAR2 directly binds or regulates Aurora B not established\", \"Connection to its nuclear scaffold functions unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Connected CCAR2 to oxidative-stress cytokine control, with its depletion activating AP-1 and IL-8 in cervical cancer cells.\",\n      \"evidence\": \"siRNA, H2O2 treatment, IL-8 ELISA/RT-PCR, AP-1 reporter, and patient tissue correlation\",\n      \"pmids\": [\"29416683\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No direct binding linking CCAR2 to the AP-1/IL-8 axis demonstrated\", \"Single lab, pathway placement inferred from reporter only\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Showed CCAR2 protein stability is controlled by a TFPI2-BRCC3 deubiquitination axis and that CCAR2 stabilizes GADD45A mRNA to inhibit HR and sensitize HCC to sorafenib.\",\n      \"evidence\": \"RNA-IP, ChIP, co-IP (TFPI2-BRCC3-CCAR2), ubiquitination assay, organoids and in vivo models\",\n      \"pmids\": [\"40765823\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct CCAR2-GADD45A mRNA binding determinants not mapped\", \"Interplay with the UBE4B degradation pathway untested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Reported a cytoplasmic CCAR2-p30 interaction in which CCAR2 supports African swine fever virus replication.\",\n      \"evidence\": \"Mass spectrometry, co-IP, co-localization, siRNA, and viral replication assay\",\n      \"pmids\": [\"39919500\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single co-IP without reciprocal/structural validation\", \"Mechanism by which CCAR2 aids replication unknown\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Identified UBE4B as an E3/E4 ligase that ubiquitinates CCAR2 for degradation, linking CCAR2 turnover to SIRT1 activity and p53 acetylation/apoptosis.\",\n      \"evidence\": \"OUT substrate screen, in vitro ubiquitination, SIRT1 activity assay, p53 acetylation western, and rescue epistasis\",\n      \"pmids\": [\"42074320\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relative contribution of CCAR2 versus direct p53 degradation by UBE4B context-dependent\", \"Signals regulating UBE4B-CCAR2 targeting unknown\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Validated the CCAR2-SIRT1 interface as a druggable target by showing distinct natural products disrupt the inhibitory complex to restore SIRT1-dependent deacetylation of p53, FXR, and pro-survival programs in liver disease.\",\n      \"evidence\": \"Affinity pull-down/MS, CETSA, docking, co-IP, substrate-acetylation assays, and liver-specific Ccar2 KO mouse models\",\n      \"pmids\": [\"41775214\", \"41825964\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Co-crystal structures of compound-bound CCAR2 not reported\", \"Selectivity over other CCAR2 functions not assessed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CCAR2's diverse activities\\u2014SIRT1 inhibition, Shieldin-dependent resection control, nuclear-receptor stabilization, chromatin remodeling, mitotic regulation, and mitochondrial survival signaling\\u2014are coordinated within a single protein, and which are governed by shared post-translational switches, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No integrated structural model linking CCAR2's domains to its multiple complexes\", \"Switching between nuclear, mitochondrial, and repair pools not mechanistically explained\", \"Whether the S1-like RNA-binding domain binds a defined RNA is unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 18, 20, 21]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 3, 12]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [13, 16]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 4, 13]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1, 10, 13]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [7]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [2, 13]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [2, 5, 13]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 3, 12]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [1, 18]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [10]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [14]}\n    ],\n    \"complexes\": [\n      \"53BP1-RIF1-Shieldin complex\",\n      \"CECR2-containing remodeling factor (CERF) complex\",\n      \"CCAR2-SIRT1 complex\",\n      \"CCAR2-Hsp60 complex\"\n    ],\n    \"partners\": [\n      \"SIRT1\",\n      \"ESR1\",\n      \"AR\",\n      \"NR1H3\",\n      \"HSPD1\",\n      \"GAPDH\",\n      \"CTNNB1\",\n      \"UBE4B\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}